Desferrithiocin polyether analogues

ABSTRACT

A relatively non-toxic desazadesferrithiocin analog having the formula: 
     
       
         
         
             
             
         
       
         
         
           
             wherein: R 1 , R 2 , R 4  and R 5  may be the same or different and may be H, straight or branched chain alkyl having up to 14 carbon atoms, e.g., methyl, ethyl, propyl and butyl, or arylalkyl wherein the aryl portion is hydrocarbyl and the alkyl portion is straight or branched chain, the arylalkyl group having up to 14 carbon atoms, R 2  optionally being alkoxy having up to 14 carbon atoms;
           R 3  is [(CH 2 ) n —O] x —[(CH 2 ) n —O] y -alkyl;   n is, independently, an integer from 1 to 8;   x is an integer from 1 to 8;   y is an integer from 0 to 8, and   R 3 O may occupy any position on the phenyl ring except the 4-position, or   
         
             a salt, hydrate or solvate thereof; and methods and compositions for treating the effects of trivalent metal, i.e., iron, overload.

RELATED APPLICATIONS

This application is a divisional of and claims priority under 35 U.S.C.§120 to U.S. application, U.S. Ser. No. 13/683,301, entitled“DESFERRITHIOCIN POLYETHER ANALOGUES,” filed Nov. 21, 2012, which is acontinuation of and claims priority under 35 U.S.C. §120 to U.S.application, U.S. Ser. No. 12/450,194, entitled “DESFERRITHIOCINPOLYETHER ANALOGUES,” filed on Dec. 14, 2009, which is a national stagefiling under 35 U.S.C. §371 of international PCT Application,PCT/US2008/003433, designating the United States and filed on Mar. 14,2008, which claims priority under 35 U.S.C. §119(e) to U.S. Provisionalapplications, U.S. Ser. No. 60/966,539, entitled “DESFERRITHIOCINPOLYETHER ANALOGUES,” filed on Mar. 15, 2007, and U.S. Ser. No.60/929,018, entitled “DESFERRITHIOCIN POLYETHER ANALOGUES,” filed onJun. 8, 2007, each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole or in part, by grant No. DK49108from the National Diabetes and Digestive and Kidney Diseases AdvisoryCouncil (NIDDK) of the National Institute of Health (NIH). TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Humans have evolved a highly efficient iron management system in whichwe absorb and excrete only about 1 mg of the metal daily; there is nomechanism for the excretion of excess iron [Brittenham, G. M. Disordersof Iron Metabolism: Iron Deficiency and Overload. In Hematology: BasicPrinciples and Practice; 3rd ed.; Hoffman, R., Benz, E. J., Shattil, S.J., Furie, B., Cohen, H. J. et al., Eds.; Churchill Livingstone: NewYork, 2000; pp 397-428]. Whether derived from transfused red blood cells[Olivieri, N. F. and Brittenham, G. M. Iron-chelating Therapy and theTreatment of Thalassemia. Blood 1997, 89, 739-761; Vichinsky, E. P.Current Issues with Blood Transfusions in Sickle Cell Disease. Semin.Hematol. 2001, 38, 14-22; Kersten, M. J., Lange, R., Smeets, M. E.,Vreugdenhil, G., Roozendaal, K. J., Lameijer, W. and Goudsmit, R.Long-Term Treatment of Transfusional Iron Overload with the Oral IronChelator Deferiprone (L1): A Dutch Multicenter Trial. Ann. Hematol.1996, 73, 247-252] or from increased absorption of dietary iron iron[Conrad, M. E.; Umbreit, J. N.; Moore, E. G. Iron Absorption andTransport. Am. J. Med. Sci. 1999, 318, 213-229; Lieu, P. T.; Heiskala,M.; Peterson, P. A; Yang, Y. The Roles of Iron in Health and Disease,Mol. Aspects Med. 2001, 22, 1-87], without effective treatment, bodyiron progressively increases with deposition in the liver, heart,pancreas, and elsewhere. Iron accumulation eventually produces (i) liverdisease that may progress to cirrhosis [Angelucci, E.; Brittenham, G.M.; McLaren, C. E.; Ripalti, M.; Baronciani, D.; Giardini, C.;Galimberti, M.; Polchi, P.; Lucarelli, G. Hepatic Iron Concentration andTotal Body Iron Stores in Thalassemia Major. N. Engl. J. Med. 2000, 343,327-331; Bonkovsky, H. L.; Lambrecht, R. W. Iron-Induced Liver Injury.Clin. Liver Dis. 2000, 4, 409429, vi-vii; Pietrangelo, A Mechanism ofIron Toxicity. Adv. Exp. Med. Biol. 2002, 509, 19-43], (ii) diabetesrelated both to iron-induced decreases in pancreatic β-cell secretionand to increases in hepatic insulin resistance [Cario, H.; Holl, R. W.;Debatin, K. M.; Kohne, E. Insulin Sensitivity and p-Cell Secretion inThalassaemia Major with Secondary Haemochromatosis: Assessment by OralGlucose Tolerance Test. Eur. J. Pediatr. 2003, 162, 139-146; Wojcik, J.P.; Speechley, M. R.; Kertesz, A E.; Chakrabarti, S.; Adams, P. C.Natural History of C282Y Homozygotes for Hemochromatosis. Can. J.Gastroenterol. 2002, 16, 297-302], and (iii) heart disease, still theleading cause of death in thalassemia major and related forms oftransfusional iron overload [Brittenham, G. M. Disorders of IronMetabolism: Iron Deficiency and Overload. In Hematology: BasicPrinciples and Practice; 3rd ed.; Hoffman, R., Benz, E. J., Shattil, S.J., Furie, B., Cohen, H. J. et aI., Eds.; Churchill Livingstone: NewYork, 2000; pp 397-428; Brittenham, G. M.; Griffith, P. M.; Nienhuis, AW.; McLaren, C. E.; Young, N. S.; Tucker, E. E.; Allen, C. J.; Farrell,D. E.; Harris, J. W. Efficacy of Deferoxamine in PreventingComplications of Iron Overload in Patients with Thalassemia Major. N.Engl. J. Med. 1994, 331, 567-573; Zurlo, M. G.; De Stefano, P.;Borgna-Pignatti, C.; Di Palma, A.; Piga, A.; Melevendi, C.; Di Gregorio,F.; Burattini, M. G.; Terzoli, S. Survival and Causes of Death inThalassaemia Major. Lancet 1989, 2, 27-30].

Although iron comprises 5% of the earth's crust, living systems havegreat difficulty in accessing and managing this vital micronutrient. Thelow solubility of Fe(III) hydroxide (K_(sp)=1×10⁻³⁹) [Raymond, K. N.;Carrano, C. J. Coordination Chemistry and Microbial Iron Transport. Ace.Chem. Res. 1979, 12, 183-190], the predominant form of the metal in thebiosphere, has led to the development of sophisticated iron storage andtransport systems in nature. Microorganisms utilize low molecularweight, virtually ferric ion-specific ligands, siderophores [Byers, B.R; Arceneaux, J. E. Microbial Iron Transport: Iron Acquisition byPathogenic Microorganisms. Met. Ions Biol. Syst. 1998,35, 37-66;Kalinowski, D. S.; Richardson, D. R. The Evolution of Iron Chelators forthe Treatment of Iron Overload Disease and Cancer. Pharmacol Rev. 2005,57, 547-583.]; higher eukaryotes tend to employ proteins to transportand store iron (e.g., transferrin and ferritin, respectively) [Bergeron,R. J. Iron: A Controlling Nutrient in Proliferative Processes. TrendsBiochem. Sci. 1986, 11, 133-136; Theil, E. c.; Huynh, B. H. FerritinMineralization: Ferroxidation and Beyond. J. Inorg. Biochem. 1997, 67,30; Ponka, P.; Beaumont, c.; Richardson, D. R. Function and Regulationof Transferrin and Ferritin, Semin. Hematol. 1998, 35, 35-54]. Inhumans, nontransferrin-bound plasma iron, a heterogeneous pool of themetal in the circulation, unmanaged iron, seems to be a principal sourceof iron-mediated organ damage.

The toxicity associated with excess iron, whether a systemic or a focalproblem, derives from its interaction with reactive oxygen species, forinstance, endogenous hydrogen peroxide (H₂O₂) [Graf, E.; Mahoney, J. R;Bryant, R. G.; Eaton, J. W. Iron-Catalyzed Hydroxyl Radical Formation.Stringent Requirement for Free Iron Coordination Site. J. Biol. Chem.1984, 259, 36203624; Halliwell, B. Free Radicals and Antioxidants: APersonal View. Nutr. Rev. 1994, 52, 253-265; Halliwell, B. Iron,Oxidative Damage, and Chelating Agents. In The Development of IronChelators for Clinical Use; Bergeron, R. J., Brittenham, G. M., Eds.;CRC: Boca Raton, 1994; pp 3356; Koppenol, W. Kinetics and Mechanism ofthe Fenton Reaction: Implications for Iron Toxicity. In Iron Chelators:New Development Strategies; Badman, D. G., Bergeron, R. J., Brittenham,G. M., Eds.; Saratoga: Ponte Vedra Beach, F L, 2000; pp 3-10]. In thepresence of Fe(II), H₂O₂ is reduced to the hydroxyl radical (HO.), avery reactive species, and HO⁻, a process known as the Fenton reaction.The Fe(III) liberated can be reduced back to Fe(II) via a variety ofbiological reductants (e.g., ascorbate), a problematic cycle. Thehydroxyl radical reacts very quickly with a variety of cellularconstituents and can initiate free radicals and radical-mediated chainprocesses that damage DNA and membranes, as well as produce carcinogens[Halliwell, B. Free Radicals and Antioxidants: A Personal View. Nutr.Rev. 1994, 52, 253-265; Babbs, C. F. Oxygen Radicals in UlcerativeColitis. Free Radic. Biol. Med. 1992, 13, 169-181; Hazen, S. L.;d'Avignon, A; Anderson, M. M.; Hsu, F. F.; Heinecke, J. W. HumanNeutrophils Employ the Myeloperoxidase-Hydrogen Peroxide-Chloride Systemto Oxidize a-Amino Acids to a Family of Reactive Aldehydes. MechanisticStudies Identifying Labile Intermediates along the Reaction Pathway. J.Biol. Chem. 1998, 273, 4997-5005]. The solution to the problem is toremove excess unmanaged iron [Bergeron, R. J.; McManis, J. S.; Weimar,W. R; Wiegand, J.; Eiler-McManis, E. Iron Chelators and TherapeuticUses. In Burger's Medicinal Chemistry; 6th ed.; Abraham, D. A, Ed.;Wiley: New York, 2003; pp 479-561].

In the majority of patients with thalassemia major or othertransfusion-dependent refractory anemias, the severity of the anemiaprecludes phlebotomy therapy as a means of removing toxic accumulationsof iron. Treatment with a chelating agent capable of sequestering ironand permitting its excretion from the body is then the only therapeuticapproach available. The iron-chelating agents now in use or underclinical evaluation [Brittenham, G. M. Iron Chelators and Iron Toxicity.Alcohol 2003, 30, 151-158] include desferrioxamine B mesylate (DFO^(a)),1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone, L1),4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid(deferasirox, ICL670A), and the desferrithiocin (DFT) analogue,(S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylicacid [deferitrin, (S)-4′-(HO)-DADFT, 1; Table 1]. Subcutaneous (sc)infusion of desferrioxamine B (DFO), a hexacoordinate hydroxamate ironchelator produced by Streptomyces pilosus [Bickel, H., Hall, G. E.,Keller-Schierlein, W., Prelog, V., Vischer, E. and Wettstein, A.Metabolic Products of Actinomycetes. XXVII. Constitutional Formula ofFerrioxamine B. Helv. Chim. Acta 1960, 43, 2129-2138], is still regardedas a credible treatment for handling transfusional iron overload[Olivieri, N. F. and Brittenham, G. M. Iron-chelating Therapy and theTreatment of Thalassemia. Blood 1997, 89, 739-761; Giardina, P. J. andGrady, R. W. Chelation Therapy in β-Thalassemia: An Optimistic Update.Semin. Hematol. 2001, 38, 360-366]. DFO is not orally active, and whenadministered sc, has a very short half-life in the body and musttherefore be given by continuous infusion over long periods of time[Olivieri, N. F. and Brittenham, G. M. Iron-chelating Therapy and theTreatment of Thalassemia. Blood 1997, 89, 739-761; Pippard, M. J.Desferrioxamine-Induced Iron Excretion in Humans. Bailliere's Clin.Haematol. 1989, 2, 323-343]. For these reasons, patient compliance is aserious problem [Olivieri, N. F. and Brittenham, G. M. Iron-chelatingTherapy and the Treatment of Thalassemia. Blood 1997, 89, 739-761;Giardina, P. J. and Grady, R. W. Chelation Therapy in β-Thalassemia: AnOptimistic Update. Semin. Hematol. 2001, 38, 360-366]. The orally activebidentate chelator, deferiprone, is licensed in Europe and some othercountries as second-line therapy to DFO [Hoffbrand, A V.; Al-Refaie, F.;Davis, B.; Siritanakatkul, N.; Jackson, B. F. A; Cochrane, J.; Prescott,E.; Wonke, B. Long-term Trial of Deferiprone in 51 Transfusion-DependentIron Overloaded Patients. Blood 1998, 91, 295-300; Olivieri, N. F.Long-term Therapy with Deferiprone. Acta Haematoi. 1996,95, 37-48;Olivieri, N. F.; Brittenham, G. M.; McLaren, C. E.; Templeton, D. M.;Cameron, R. G.; McClelland, R. A; Burt, A D.; Fleming, K. A Long-TermSafety and Effectiveness of Iron-Chelation Therapy with Deferiprone forThalassemia Major. N. Engi. J. Med. 1998, 339, 417-423; Richardson, D.R. The Controversial Role of Deferiprone in the Treatment ofThalassemia. J. Lab. Clin. Med. 2001, 137, 324-329]. Unfortunately,although it is orally active, it is less efficient than sc DFO atremoving iron. Whereas the orally active tridentate chelator deferasiroxhas now been approved by the FDA, it did not demonstrate non-inferiorityto DFO. Furthermore, it apparently has a somewhat narrow therapeuticwindow, owing to potential nephrotoxicity, noted in animals during thepreclinical toxicity studies [Nisbet-Brown, E.; Olivieri, N. F.;Giardina, P. J.; Grady, R. W.; Neufeld, E. J.; Sechaud, R; Krebs-Brown,A J.; Anderson, J. R; Alberti, D.; Sizer, K. c.; Nathan, D. G.Effectiveness and Safety of ICL670 in Iron-Loaded Patients withThalassaemia: a Randomised, Double-Blind, Placebo-Controlled,Dose-Escalation Trial. Lancet 2003, 361, 1597-1602; Galanello, R; Piga,A; Alberti, D.; Rouan, M.-C.; Bigler, H.; Sechaud, R. Safety,Tolerability, and Pharmacokinetics of ICL670, a New Orally ActiveIron-Chelating Agent in Patients with Transfusion-Dependent IronOverload Due to Thalassemia. J. Clin. Pharmacol. 2003, 43, 565-572;Cappellini, M. D. Iron-chelating therapy with the new oral agent ICL670(Exjade). Best Pract Res Clin Haematol 2005, 18, 289-298]. In addition,Novartis has recently (April, 2007) updated the prescribing informationfor deferasirox: “Cases of acute renal failure, some with a fataloutcome, have been reported following the postmarketing use of Exjade®(deferasirox). Most of the fatalities occurred in patients with multipleco-morbidities and who were in advanced stages of their hematologicaldisorders” [Exjade Prescribing Information,http://www.pharma.us.novartis.com/product/pi/pdf/exjade.pdf (accessedMay 2007)]. Finally, ligand 1 is an orally active tridentate DFTanalogue now in phase 1/II trials in patients. Although the preclinicaltoxicity profile of 1 was relatively benign, that is, no geno- orreproductive toxicity and only mild nephrotoxicity at high doses, theclinical results remain to be elucidated.

It is an object of the present invention to provide noveldesferrithiocin analogues useful for the treatment of iron overload inmammals and the diseases associated therewith.

SUMMARY OF THE INVENTION

The above and other objects are realized by the present invention, oneembodiment of which relates to relatively non-toxicdesazadesferrithiocin analogs having the formula:

-   -   wherein: R₁, R₂, R₄ and R₅ may be the same or different and may        be H, straight or branched chain alkyl having up to 14 carbon        atoms, e.g., methyl, ethyl, propyl and butyl, or arylalkyl        wherein the aryl portion is hydrocarbyl and the alkyl portion is        straight or branched chain, the arylalkyl group having up to 14        carbon atoms, R₂ R₂ optionally being alkoxy having up to 14        carbon atoms;    -   R₃ is [(CH₂)n-O]_(x)—[(CH₂)n-O]_(y)-alkyl;    -   n is, independently, an integer from 1 to 8;    -   x is an integer from 1 to 8;    -   y is an integer from 0 to 8, and    -   R₃O may occupy any position on the phenyl ring except    -   the 4-position, or a salt, hydrate or solvate thereof.

An additional embodiment of the invention relates to a method oftreating a pathological condition responsive to chelation orsequestration of a trivalent metal in a subject comprising administeringto the subject a therapeutically effective amount of an analog describedabove.

A still further embodiment of the invention relates to a pharmaceuticalcomposition for treating a pathological condition responsive tochelation or sequestration of a trivalent metal comprising an effectiveamount of at least one analog described above and a pharmaceuticallyacceptable carrier therefore.

Another embodiment of the invention relates to an article of manufacturecomprising packaging material and a pharmaceutical agent containedwithin said packaging material, wherein said pharmaceutical agent iseffective for the treatment of a subject suffering from trivalent metaloverload, and wherein said packaging material comprises a label whichindicates that said pharmaceutical agent can be used for amelioratingthe symptoms associated with trivalent metal overload, and wherein saidpharmaceutical agent is an analog described above.

There has thus been outlined, rather broadly, the more importantfeatures of the invention in order that the detailed description thereofthat follows may be better understood, and in order that the presentcontribution to the art may be better appreciated. There are, of course,additional features of the invention that will be described furtherhereinafter. Indeed, it is to be understood that both the foregoinggeneral description and the following detailed description are exemplaryand explanatory and are intended to provide further explanation of theinvention as claimed.

In this respect, before explaining at least one embodiment of theinvention in detail, it is to be understood that the invention is notlimited in its application to the details of construction and to thearrangements of the components set forth in the following description orillustrated in the drawings. The invention is capable of otherembodiments and of being practiced and carried out in various ways.Also, it is to be understood that the phraseology and terminologyemployed herein are for the purpose of description and should not beregarded as limiting.

As such, those skilled in the art will appreciate that the conceptionupon which this disclosure is based may readily be utilized as a basisfor the designing of other structures, methods and systems for carryingout the several purposes of the present invention. It is important,therefore, that equivalent constructions insofar as they do not departfrom the spirit and scope of the present invention, are included in thepresent invention.

The accompanying drawings are included to provide a furtherunderstanding of the invention and are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description serve to explain theprincipals of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. ¹H resonances and pertinent homonuclear NOE correlations for(S)-3′-(HO)-DADFT-PE (9); the percent NOE is indicated next to thedotted lines.

FIG. 2. ¹H resonances and pertinent homonuclear NOE correlations for(S)-5′-(HO)-DADFT-PE-iPrE (12); the percent NOE is indicated next to thedotted lines.

FIG. 3. Biliary ferrokinetics of rats treated with DADFT analogues 1, 3,6 and 9 given po at a dose of 300 μmol/kg. The iron clearance (y-axis)is reported as μg of iron per kg body weight.

FIG. 4. Tissue distribution in plasma, kidney, liver, heart and pancreasof rats treated with DADFT analogues 1, 3, 6 and 9 given sc at a dose of300 μmol/kg. The concentrations (y-axis) are reported as μM (plasma) oras nmol compound per g wet weight of tissue. For all time points, n=3.

FIG. 5—Scheme 1. Synthesis of (S)-5′-(OH)-DADFT-PE (6) and(S)-3′-(OH)-DADFT-PE (9).

FIG. 6—Schemes 1 & 2. Syntheses of(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-4′-(HO)-DADFT-PE] and[(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-3′-(HO)-DADFT-PE], respectively

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the discovery thatdesazadesferrithiocin analogs as described above are very effectiverelatively non-toxic chelators of trivalent metals, particularly iron,in mammals.

More particularly, the present invention is predicated on the discoverythat introducing polyether groups at various positions of thedesazadesferrithiocin (DADFT) aromatic ring greatly enhances the ironclearance and organ distribution properties of the resulting analogues.Three DADFT polyethers are evaluated:(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-4′-(HO)-DADFT-PE, 3],S)-4,5-dihydro-2-[2-hydroxy-5-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-5′-(HO)-DADFT-PE, 6], and(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-3′-(HO)-DADFT-PE, 9]. The iron-clearing efficiency (ICE) inrodents and primates is shown to be very sensitive to which positionalisomer is evaluated, as is the organ distribution in rodents. Thepolyethers had uniformly higher ICEs than their corresponding parentligands in rodents, consistent with in vivo ligand-serum albumin bindingstudies. Ligand 9 is the most active polyether analogue in rodents andis also very effective in primates, suggesting a higher index of successin humans. In addition, this analogue is also shown to clear more ironin the urine of the primates than many of the other chelators. If thistrend was also observed in patients, performance of iron-balance studiesin a clinical setting would be much easier.

Ligand 1 is an orally active tridentate DFT analogue now in Phase VIItrials in patients. Although the preclinical toxicity profile of 1 wasrelatively benign, i.e., no geno- or reproductive toxicity and only mildnephrotoxicity at high doses, the clinical results remain to beelucidated. Previous studies revealed that within a family ofdesferrithiocin analogues the more lipophilic chelators have betteriron-clearing efficiency, that is, the larger the log P_(app) value ofthe compound, the better the iron-clearing efficiency (ICE) [Bergeron,R. J., Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao, H., Bharti,N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783; Bergeron, R. J., Wiegand, J., McManis, J. S., Bussenius, J.,Smith, R. E. and Weimar, W. R. Methoxylation of DesazadesferrithiocinAnalogues: Enhanced Iron Clearing Efficiency. J. Med. Chem. 2003, 46,1470-1477; Bergeron, R. J., Wiegand, J., McManis, J. S. and Bharti, N.The Design, Synthesis, and Evaluation of Organ-Specific Iron Chelators.J. Med. Chem. 2006, 49, 7032-7043]. There also exists a second, albeitsomewhat disturbing relationship: in all sets of ligands, the morelipophilic chelator is always the more toxic [Bergeron, R. J.; Wiegand,J.; McManis, J. S.; Vinson, J. R. T.; Yao, H.; Bharti, N.; Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. A previous investigation focused on the design of ligandsthat balance the lipophilicity/toxicity relationship while iron-clearingefficiency is maintained. The study began with the observation that(S)-4,5-dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-methyl-4-thiazolecarboxylicacid [(S)-4′-(CH₃O)-DADFT, 2], a 4′-methyl-ether, had excellentiron-clearing efficiency in both rodents and primates; however, it wasunacceptably toxic [Bergeron, R. J., Wiegand, J., McManis, J. S.,Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783; Bergeron, R. J., Wiegand, J., McManis, J. S., Bussenius, J.,Smith, R. E. and Weimar, W. R. Methoxylation of DesazadesferrithiocinAnalogues: Enhanced Iron Clearing Efficiency. J. Med. Chem. 2003, 46,1470-1477]. Nevertheless, this established that alkylation of the4′-(HO) functionality of (S)-4′-(HO)-DADFT (1) was compatible with theiron-clearing function. On the basis of these observations, a lesslipophilic, more water-soluble ligand than 2 was assembled, thepolyether(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-4′-(HO)-DADFT-PE, 3] [Bergeron, R. J., Wiegand, J., McManis,J. S., Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783].

When 1 was 4′-methoxylated to provide 2, the ICE in a rodent model afteroral (po) administration increased substantially from 1.1±0.8% to6.6±2.8% (p<0.02) [Bergeron, R. J., Wiegand, J., McManis, J. S. andBharti, N. The Design, Synthesis, and Evaluation of Organ-Specific IronChelators. J. Med. Chem. 2006, 49, 7032-7043]. The polyether (3), inwhich a 3,6,9-trioxadecyl group was fixed to the 4′-(HO) of 1, alsoperformed well, with an ICE of 5.5±1.9% when administered po (p<0.003vs 1) [Bergeron, R. J., Wiegand, J., McManis, J. S., Vinson, J. R. T.,Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. The efficiency of 1 given po to iron-loaded primates was16.8±7.2% [Bergeron, R. J., Wiegand, J., McManis, J. S. and Bharti, N.The Design, Synthesis, and Evaluation of Organ-Specific Iron Chelators.J. Med. Chem. 2006, 49, 7032-7043] while the ICE of the 4′-(CH₃O)analogue (2) given po was 24.4±10.8% [Bergeron, R. J., Wiegand, J.,McManis, J. S., Bussenius, J., Smith, R. E. and Weimar, W. R.Methoxylation of Desazadesferrithiocin Analogues: Enhanced Iron ClearingEfficiency. J. Med. Chem. 2003, 46, 1470-1477]. The correspondingpolyether (3) given po performed very well in primates with anefficiency of 25.4±7.4% [Bergeron, R. J., Wiegand, J., McManis, J. S.,Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783].

Earlier studies carried out in rodents clearly demonstrated thepolyether (S)-4′-(HO)-DADFT-PE (3) to be less nephrotoxic than thecorresponding (S)-4′-(CH₃O)-DADFT analogue (2) or the parent drug(S)-4′-(HO)-DADFT (1) [Bergeron, R. J., Wiegand, J., McManis, J. S.,Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. In an attempt to understand this difference in toxicity, thetissue levels of 3 and 1 in the liver, kidney, pancreas, and heart ofrodents given a single sc 300 μmol/kg dose of the chelators weremeasured 2, 4, 6, and 8 h after exposure [Bergeron, R. J., Wiegand, J.,McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. There were two notable observations. At each time point, thelevel of the polyether 3 in the liver was much higher than that of theparent drug 1. In the kidney, the polyether concentration was lower thanthe parent at 2 h and similar at later time points [Bergeron, R. J.,Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N. andRocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. This seemed consistent with the reduced nephrotoxicity. R.Furthermore, in an experiment in which 1 and 3 were given po to the ratstwice daily at a dose of 237 μmol/kg/dose (474 μmol/kg/day) for 7 days[Bergeron, R. J., Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao,H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783], under light microscopy, the proximal tubules of kidneys fromthe polyether (3)-treated rodents were indistinguishable from those ofthe control animals; the distal tubules presented with occasionalvacuolization but were otherwise normal [Bergeron, R. J., Wiegand, J.,McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. However, animals treated with 1 showed regional, moderate tosevere vacuolization in the proximal tubules, a loss of the brushborder, and tubular extrusions toward the lumen; the distal tubulesshowed moderate to severe vacuolization. These findings, coupled withthe increased ICE of the polyether 3, compelled us to pursue furtherstudies on additional polyethers and evaluate drug tissue levels atadditional (earlier) time points.

Desferrithiocin,(S)-4,5-dihydro-2-(3-hydroxy-2-pyridinyl)-4-methyl-4-thiazolecarboxylicacid (DFT), is a tridentate siderophore siderophore [Naegeli, H.-D.;Zahner, H. Metabolites of Microorganisms. Part 193. Ferrithiocin. Helv.Chim. Acta 1980, 63, 1400-1406] that forms a stable 2:1 complex withFe(III); the cumulative formation constant is 4×10²⁹ M⁻¹ [Hahn, F. E.;McMurry, T. J.; Hugi, A; Raymond, K. N. Coordination Chemistry ofMicrobial Iron Transport. 42. Structural and SpectroscopicCharacterization of Diastereomeric Cr(III) and Co(III) Complexes ofDesferriferrithiocin. J. Am. Chem. Soc. 1990, 112, 1854-1860; Anderegg,G.; Raber, M. Metal Complex Formation of a New SiderophoreDesferrithiocin and of Three Related Ligands. J. Chern. Soc. Chern.Cornrnun. 1990, 1194-1196]. It performed well when given orally (po) inboth the bile duct-cannulated rodent model (ICE, 5.5%) [Bergeron, R. J.;Wiegand, J.; Dionis, J. B.; Egli-Karmakka, M.; Frei, J.; Huxley-Tencer,A.; Peter, H. H. Evaluation of Desferrithiocin and Its SyntheticAnalogues as Orally Effective Iron Chelators. J. Med. Chern. 1991, 34,2072-2078] and in the iron-overloaded Cebus apella primate (ICE, 16%)[Bergeron, R. J.; Streiff, R. R; Creary, E. A; Daniels, R. D., Jr.;King, W.; Luchetta, G.; Wiegand, J.; Moerker, T.; Peter, H. H. AComparative Study of the Iron-Clearing Properties of DesferrithiocinAnalogues with DFO in a Cebus Monkey Model. Blood 1993, 81, 21662173;Bergeron, R. J.; Streiff, R. R; Wiegand, J.; Vinson, J. R. T.; Luchetta,G.; Evans, K. M.; Peter, H.; Jenny, H.-B. A Comparative Evaluation ofIron Clearance Models. Ann. N. Y. Acad. Sci. 1990, 612, 378-393].Unfortunately, DFT is severely nephrotoxic [Bergeron, R. J., Streiff, R.R., Creary, E. A., Daniels, R. D., Jr., King, W., Luchetta, G., Wiegand,J., Moerker, T. and Peter, H. H. A Comparative Study of theIron-Clearing Properties of Desferrithiocin Analogues withDesferrioxamine B in a Cebus Monkey Model. Blood 1993, 81, 2166-2173].Nevertheless, the outstanding oral activity spurred a structure-activitystudy to identify an orally active and safe DFT analogue. The initialgoal was to define the minimal structural platform compatible with ironclearance on po administration [Bergeron, R. J., Streiff, R. R., Creary,E. A., Daniels, R. D., Jr., King, W., Luchetta, G., Wiegand, J.,Moerker, T. and Peter, H. H. A Comparative Study of the Iron-ClearingProperties of Desferrithiocin Analogues with Desferrioxamine B in aCebus Monkey Model. Blood 1993, 81, 2166-2173]′[Bergeron, R. J.,Wiegand, J., Dionis, J. B., Egli-Karmakka, M., Frei, J., Huxley-Tencer,A. and Peter, H. H. Evaluation of Desferrithiocin and Its SyntheticAnalogues as Orally Effective Iron Chelators. J. Med. Chem. 1991, 34,2072-2078]. This was followed by a series of structure-activity studiesaimed at developing a DFT analogue with good oral iron-clearing activityand an acceptable toxicity profile [Bergeron, R. J., Wiegand, J.,McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783; Bergeron, R. J., Wiegand, J., McManis, J. S., McCosar, B. H.,Weimar, W. R., Brittenham, G. M. and Smith, R. E. Effects of C-4Stereochemistry and C-4′ Hydroxylation on the Iron Clearing Efficiencyand Toxicity of Desferrithiocin Analogues. J. Med. Chem. 1999, 42,2432-2440]. The outcome was (S)-4′-(HO)-DADFT (1), now in clinicaltrials. However, animal studies suggested that even in this system, thedose-limiting toxicity would likely be nephrotoxicity [Bergeron, R. J.,Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N. andRocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. We next discovered that fixing a polyether to the4′-position leading to (S)-4′-(HO)-DADFT-PE (3) profoundly reducednephrotoxicity. The reduction in proximal tubule damage seemedconsistent with the reduced level of 3 in the kidney relative to theparent ligand 1 at 2 h [Bergeron, R. J., Wiegand, J., McManis, J. S.,Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783].

It was decided to better understand the role the polyether fragmentplays from a positional isomer standpoint; the design strategies arebased on comparative issues. Three questions are addressed: How doesaltering the position of the polyether in the aromatic ring affect (1)iron-clearing efficiency in rodents, (2) iron-clearing efficiency inprimates, and (3) tissue distribution in rodents? With this information,we will decide how best to conduct possible further and protractedtoxicity trials in rodents

A platform,(S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-thiazolecarboxylic acid(DADFT) is evaluated in this study. In each instance, a singlesubstituent, hydroxy, methoxy, or 3,6,9-trioxadecyloxy was added to the3′ (7-9), 4′ (1-3) or 5′ (4-6) positions of the aromatic ring. In eachinstance, the iron-clearance data is presented in both rodents andprimates, along with log P_(app) numbers (Tables 1 and 2). Historicaldata are included [Bergeron, R. J.; Wiegand, J.; McManis, J. S.; Bharti,N. The Design, Synthesis, and Evaluation of Organ-Specific IronChelators. J. Med. Chem. 2006, 49, 7032-7043; Bergeron, R. J.; Wiegand,J.; McManis, J. S.; Vinson, J. R. T.; Yao, H.; Bharti, N.; Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783; Bergeron, R. J.; Wiegand, J.; McManis, J. S.; McCosar, B. H.;Weimar, W. R; Brittenham, G. M.; Smith, R. E. Effects of C-4Stereochemistry and C-4′ Hydroxylation on the Iron Clearing Efficiencyand Toxicity of Desferrithiocin Analogues. J. Med. Chem. 1999, 42,2432-2440; Bergeron, R. J.; Wiegand, J.; McManis, J. S.; Bussenius, J.;Smith, R. E.; Weimar, W. R. Methoxylation of DesazadesferrithiocinAnalogues: Enhanced Iron Clearing Efficiency. J. Med. Chem. 2003, 46,1470-1477; Bergeron, R. J.; Wiegand, J.; McManis, J. S.; Weimar, W. R;Park, J.-H.; Eiler-McManis, E.; Bergeron, J.; Brittenham, G. M.Partition-Variant Desferrithiocin Analogues: Organ Targeting andIncreased Iron Clearance. J. Med. Chem. 2005, 48, 821-831]. Discussionof organ distribution of the ligands in rodents is limited to(S)-4′-(HO)-DADFT (1) and the three trioxadecyloxy compounds 3, 6 and 9(FIG. 4). Organ distribution data for the non-polyethers 2, 4 and 5 canbe found in a previous publication [Bergeron, R. J.; Wiegand, J.;McManis, J. S.; Bharti, N. The Design, Synthesis, and Evaluation ofOrgan-Specific Iron Chelators. J. Med. Chem. 2006, 49, 7032-7043].Supporting Information Available: Elemental analytical data forsynthesized compounds.

The syntheses of(S)-4,5-dihydro-2-[2-hydroxy-5-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-5′-(HO)-DADFT-PE, 6] and(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-3′-(HO)-DADFT-PE (9)] were achieved by first converting(S)-2-(2,5-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylicacid [(S)-5′-(HO)-DADFT, 4] and(S)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylicacid [(S)-3′-(HO)-DADFT, 7] to their isopropyl ester 10 and ethyl ester11, respectively (Scheme 1). (S)-5′-(HO)-DADFT (4) was converted to itsisopropyl ester 10 in quantitative yield by alkylation with2-iodopropane (1.8 equiv) in DMF in the presence of N,N-diisopropylethylamine (1.8 equiv). The ethyl ester of(S)-3′-(HO)-DADFT, 11, was accessed by alkylation of 7 with iodoethane(1.8 equiv) and N,N-diisopropylethylamine (1.8 equiv) in DMF. Compounds10 and 11 were then alkylated at the 5′-hydroxyl and 3′-hydroxyl usingan equimolar amount of tri(ethylene glycol) monomethyl ether underMitsunobu conditions [diisopropyl azodicarboxylate (1.19 equiv) andtriphenylphosphine (1.23 equiv) in THF], providing(S)-5′-(HO)-DADFT-PE-iPrE (12) and (S)-3′-(HO)-DADFT-PE-EE (13) in 52and 25% yields, respectively. Hydrolysis of isopropyl and ethyl esterwith 50% NaOH in methanol followed by acidification with 2N HClfurnished (S)-5′-(HO)-DADFT-PE (6, 97%) and (S)-3′-(HO)-DADFT-PE (9) in60% yield.

To demonstrate that the polyether chain of 9 was indeed fixed to the3′-position and not to its 2′-hydroxyl, proton nuclear Overhauser effect(NOE) difference spectra were acquired and the results are shown inFIG. 1. Low-power saturation of the resonance at 4.28 ppm, assigned tothe protons of the polyether's methylene (g) most proximate to thearomatic residue, enhanced the signal for the adjacent methylene (e) at3.94 ppm by 6%, while a single aromatic signal at 7.30 ppm (i) alsoshowed a significant enhancement of 11%. These observations areconsistent with the structure for 9.

Proton NOE difference spectroscopy was also used to verify thatalkylation of the polyether chain occurred at the 5′-position in 12 andnot at the more sterically hindered 2′-hydroxyl; these results are shownin FIG. 2. Irradiation of the signal at 4.10 ppm, assigned to methylene(h) in the polyether chain, enhanced the neighboring methylene (f)resonance at 3.84 ppm by 6%, and two aromatic signals at 6.94 ppm (j)and 7.01 ppm (k) showed significant enhancements of 13% and 7%respectively. These enhancements indicate that the structure for 12 iscorrect as shown and, thus, the resulting hydrolysis product is indeed6.

Chelator-Induced Iron Clearance in Non-Iron-Overloaded Rodents. Wepreviously demonstrated that in the (S)-4′-(HO)-DADFT series,(S)-4′-(HO)-DADFT (1), (S)-4′-(CH₃O)-DADFT (2), and (S)-4′-(HO)-DADFT-PE(3), both the methoxy ligand (2; ICE 6.6±2.8%) and the polyether (3; ICE5.5±1.9%) were more efficient iron chelators than the parent ligand 1;ICE 1.1±0.8% (p<0.02 vs 2 and p<0.003 vs 3), respectively (Table 1)[Bergeron, R. J., Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao,H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. Recall that iron-clearing efficiency (ICE) is defined as(net iron excretion)/(total iron-binding capacity of the chelator),expressed as a percent. The relative ICE values of compounds 1 and 2were in keeping with their log P_(app) values: the more lipophilic, thelarger log P_(app), the more efficient the chelator. This was not thecase with the polyether analogue 3; it was much more active than its logP_(app) would have predicted. However, the ICE trend was in keeping withthe biliary ferrokinetics (FIG. 3) and liver concentration of thechelators, for example, 3>1 (FIG. 4).

With the (S)-5′-(HO)-DADFT series (Table 1), (S)-5′-(HO)-DADFT (4),(S)-4,5-dihydro-2-(2-hydroxy-5-methoxyphenyl)-4-methyl-4-thiazolecarboxylicacid [(S)-5′-(CH₃O)-DADFT, 5], and (S)-5′-(HO)-DADFT-PE (6), both themethoxy analogue 5 (ICE 6.3±1.2%) and the polyether 6 (ICE 8.0±1.8%)were more efficient iron chelators than the parent ligand 4 (ICE1.0±0.9%, p<0.001 vs 5 and p<0.005 vs 6, respectively). Again, therelative ICEs of 4 versus 5 were in keeping with the log P_(app) andliver concentrations [Bergeron, R. J., Wiegand, J., McManis, J. S. andBharti, N. The Design, Synthesis, and Evaluation of Organ-Specific IronChelators. J. Med. Chem. 2006, 49, 7032-7043]. Although liverconcentration (FIG. 4) was a good indicator of the ICE of polyether 6,relative to 4 [Bergeron, R. J., Wiegand, J., McManis, J. S. and Bharti,N. The Design, Synthesis, and Evaluation of Organ-Specific IronChelators. J. Med. Chem. 2006, 49, 7032-7043], log P_(app) was not. Itis notable that in both the (S)-5′-(HO)-DADFT and the (S)-4′-(HO)-DADFTseries, the corresponding HO—, CH₃O— and polyether ligands have similarICE values and similar iron-clearance distribution in the urine and bile(Table 1).

The ICEs of the (S)-3′-(HO)-DADFT set of compounds 7-9 are verydifferent than the 4′-(HO) and 5′-(HO) series. The ligands are, as afamily, more efficient at clearing iron (Table 1). Again, although therelative iron-clearing efficiencies are predicted by the log P_(app)values for the 3′-(HO) and 3′-(CH₃O) compounds, the ICE for the3′-polyether (9) is not. What is most relevant in this instance is theprofound difference in ICE between the 3′-polyether (9) and the4′-polyether (3); the 3′-ligand (9) is nearly 200% more effective(10.6±4.4% vs 5.5±1.9%, p<0.05). The ICE of 9 is also greater than thatof the 5′-polyether 6, (10.6±4.4% vs 8.0±1.8%, respectively), but theincrease is slightly less than significant (p=0.06). The modes of ironexcretion, urine versus bile, are similar.

The biliary ferrokinetics of the parent 1 and the three polyethers 3, 6and 9 (FIG. 3) show that the iron clearance (μg/kg) of ligand 1 peaks at3 h postdrug and never exceeds 68 μg/kg. The iron excretion induced bythe 4′- and 5′-polyethers (3 and 6) also peak at 3 h, but at much higherlevels, 183 and 388 μg/kg, respectively (p<0.001 for 1 vs 3 or 6). Thebiliary iron content of 3′-polyether 9 treated animals is greatest at 6h, 287 μg/kg. In addition, while the biliary iron clearance for 1, 3 and6 have returned to baseline levels by 15 h, the 3′-polyether (9) remainswell above this until >30 h (data not shown). The delayed peak in ironexcretion and duration of activity of 9 are also reflected in the tissuedistribution studies (FIG. 4), discussed below.

Chelator-Induced Iron Clearance in Iron-Overloaded Cebus apellaPrimates. The iron-clearance data for all three sets of ligands arepresented (Table 2). In the case of the (S)-4′-(HO)-DADFT series inprimates, while the mean ICE values for (S)-4′-(HO)-DADFT (1) and(S)-4′-(CH₃O)-DADFT (2) suggest a correlation with log P_(app), forexample, the ICE of the more lipophilic analogue 2 (24.4±10.8%)[Bergeron, R. J., Wiegand, J., McManis, J. S., Bussenius, J., Smith, R.E. and Weimar, W. R. Methoxylation of Desazadesferrithiocin Analogues:Enhanced Iron Clearing Efficiency. J. Med. Chem. 2003, 46, 1470-1477]>1(16.8±7.2%) [Bergeron, R. J., Wiegand, J., McManis, J. S., McCosar, B.H., Weimar, W. R., Brittenham, G. M. and Smith, R. E. Effects of C-4Stereochemistry and C-4′ Hydroxylation on the Iron Clearing Efficiencyand Toxicity of Desferrithiocin Analogues. J. Med. Chem. 1999, 42,2432-2440], the increase is not significant. Although(S)-4′-(HO)-DADFT-PE (3) is the least lipophilic chelator in the 1-3series, it is just as efficient (ICE, 25.4±7.4%) [Bergeron, R. J.,Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N. andRocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783] as analogue 2 and is slightly more effective than the parent1, although the increase was not quite significant (p=0.06).

In the case of the (S)-5′-(HO)-DADFT analogues 4-6, the ligands' ICEtrend correlates well with log P_(app). The ICE of the most lipophilicligand 5 (18.9±2.3%) is more than twice as efficient as the leastlipophilic analogue 6 (ICE 8.1±2.8%, p<0.001); 5 is also more efficientthan chelator 4, ICE 12.6±3.0% (p<0.01) [Bergeron, R. J., Wiegand, J.,McManis, J. S. and Bharti, N. The Design, Synthesis, and Evaluation ofOrgan-Specific Iron Chelators. J. Med. Chem. 2006, 49, 7032-7043]. Withthe (S)-3′-(HO)-DADFT analogues 7-9, while there are clear differencesin log P_(app), the ICEs of the three ligands are all within error ofeach other (Table 2).

A final, comparative note concerning the polyether ligands relates tothe biliary versus urinary metal excretion. In the rodent model, thenumbers are generally similar, with nearly all of the iron excreted inthe bile (Table 1). This is not the case with the primates; a muchlarger fraction of the iron is found in the urine (Table 2). The mostnotable cases are (S)-5′-(HO)-DADFT-PE (6) with 56/44 feces/urine ratioand (S)-3′-(HO)-DADFT-PE (9) with 72/28 feces/urine ratio in primates.In rodents, these numbers are 98/2 and 95/5, respectively.

Iron Clearance Performance Ratio in Primates versus Rodents. Theperformance ratio (PR), defined as the meanICE_(primates)/ICE_(rodents), is noteworthy (Table 3). At first glance,it does not seem surprising that the ligands uniformly perform better inthe iron-overloaded primates than in the non-iron-overloaded rats(Tables 1 and 2). The mean ICE for the primate group can be comparedwith the mean ICE of the rodent group (Table 3). Although the standarddeviations for the two species are not equivalent for 1-5 and 7-9, thisis not a concern because the intervals containing the means do notinteract. This is not the case with the 5′-polyether (6), whose ICE isvirtually identical in the two species. The largest differences inperformance ratios generally unfold with the parent ligands 1, 4, and 7.However, the fact that the ratios change so profoundly within sets(i.e., 1 vs 2 and 3, 4 vs 5 and 6, and 7 vs 8 and 9) suggests that thedifference in ICE in primates versus rodents is not based entirely onthe fact the monkeys are iron-overloaded, while the rodents are not.

The Possible Impact of Ligand-Albumin Binding on ICE. In an attempt tounderstand the differences in ICE between the parent ligands 1, 4, and 7and their analogues in the rodent model, we conducted a series ofexperiments focused on ligand-albumin binding. We elected to focus on adrug that is under clinical trials with Genzyme, (S)-4′-(HO)-DADFT (1).Recall the corresponding polyether (3) performed significantly better inthe rodent (ICE 5.5±1.9% vs 1.1±0.8% for 3 and 1, respectively)[Bergeron, R. J., Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao,H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. A series of comparative experiments in rats focused ondisplacing (S)-4′-(HO)-DADFT (1) and (S)-4′-(HO)-DADFT-PE (3) from serumalbumin binding sites were carried out.

Benzoic acid has been well established as a ligand that will displacedrugs from both sites in human serum albumin [Ostergaard, J., Schou, C.,Larsen, C. and Heegaard, N. H. Evaluation of capillaryelectrophoresis-frontal analysis for the study of low molecular weightdrug-human serum albumin interactions. Electrophoresis 2002, 23,2842-2853]. We elected to evaluate the impact of treating bileduct-cannulated rodents with sodium benzoate to displace any chelatorpotentially bound to Sudlow types I and II albumin binding sites[Ostergaard, J., Schou, C., Larsen, C. and Heegaard, N. H. Evaluation ofcapillary electrophoresis-frontal analysis for the study of lowmolecular weight drug-human serum albumin interactions. Electrophoresis2002, 23, 2842-2853]. Five experiments were carried out (Table 4).Rodents were given (i) sodium benzoate dissolved in distilled water at250 mg/kg/dose sc times six doses, (ii) (S)-4′-(HO)-DADFT (1) po at 300μmol/kg, (iii) 1 given po at 300 μmol/kg plus sodium benzoate (250mg/kg/dose). The sodium benzoate was given 0.5 h pre-1 and hourlythereafter for five additional doses, (iv) (S)-4′-(HO)-DADFT-PE (3)administered po at 300 μmol/kg, or (v) 3 dosed at 300 μmol/kg po plussodium benzoate (250 mg/kg/dose). The sodium benzoate was again given0.5 h pre-3 and hourly thereafter for five additional doses. The results(Table 4) indicate that the sc administration of sodium benzoate itselfdoes not induce the clearance of any iron. However, when sodium benzoateis administered to the rodents as described above in addition to 1,there is a 10.9-fold increase in the ICE, from 1.1±0.8% to 12.0±2.6%(p<0.001). Under the same conditions, the ICE of (S)-4′-(HO)-DADFT-PE(3) also increases, but by a much smaller magnitude, from 5.5±1.9% to8.8±2.4% (p<0.05), a 1.6-fold increase. Lower dosing of sodium benzoatehad a lesser effect on increasing the ICE of 1 (data not shown). Thesedata are consistent with the idea that the difference in ICE in rodentsbetween parent ligands and the corresponding polyethers may well bedependent on ligand-albumin binding differences. The data may also beconsistent with the difference in performance ratios in primates versusrodents, that is, the ligands may uniformly bind less tightly to primatealbumin than to rodent albumin.

Chelator Tissue Distribution in Rodents. Two issues were addressedregarding moving the 3,6,9-trioxadecyloxy (polyether) group around theDADFT aromatic ring—the impact on ICE and the effect on tissuedistribution. These assessments represent the first step in identifyingwhich, if any, additional DADFT polyethers should be moved forward intoprotracted toxicity trials in rodents.

The current study clearly indicates that moving the polyether from the4′- to the 3′- or 5′-position of the aromatic ring of DADFT can have aprofound effect on ICE (Tables 1 and 2) and tissue distribution (FIG. 4)of the resulting ligands. In the kidney (FIG. 4) at the 0.5 h timepoint, the 5′-polyether (6) achieved the highest concentration (643±92nmol/g wet weight), followed by the 4′-polyether (3; 368±74 nmol/g wetweight, p<0.01 vs 6) and 3′-polyether (9; 280±26 nmol/g wet weight,p<0.01 vs 6). Interestingly, at this time point, the concentration ofthe 4′-polyether (3) and the parent (1; 361±56 nmol/g wet weight) werenearly identical (p>0.05). At 1 h, again the 5′-ligand (6) was mostconcentrated (435±111 nmol/g wet weight), with the 3′-chelator (9) and4′-chelator (3) achieving very similar levels (259±35 and 252±10 nmol/gwet weight, respectively). The parent drug 1 was the least concentrated(179±4 nmol/g wet weight). At 2 h, the relative kidney levels are indeeddifferent. Again, the 5′-ligand (6) was most concentrated (321±20 nmol/gwet weight)>>9; 145±27 nmol/g wet weight>>3; 41±3 nmol/g wet weight(p<0.001 for 6 vs 9 or 3). At 4 h, the order was now 6 (116±65 nmol/gwet weight)≈9 (90±7 nmol/g wet weight)>3 (34±13 nmol/g wet weight)≈1(27±7 nmol/g wet weight). Recall that previous studies demonstrated the4′-polyether (3) to be much less nephrotoxic than the parent drug(S)-4′-(HO)-DADFT (1) [Bergeron, R. J., Wiegand, J., McManis, J. S.,Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. This was consistent with the relative tissue levels at the 2h time point: the 4′-polyether concentration 3 was much lower than theparent 1 [Bergeron, R. J., Wiegand, J., McManis, J. S., Vinson, J. R.T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783] However, renal concentration data derived from time pointstaken earlier than 2 h are not consistent with the idea that reducednephrotoxicity of the 4′-polyether (3) relative to the parent (1) can beexplained simply by lower kidney chelator levels.

At 0.5 h, the liver concentrations of the three polyether ligands (FIG.4) follow the same relative order as seen in the kidney, 6 (483±85nmol/g wet weight)>3 (339±35 nmol/g wet weight)>9 (242±38 nmol/g wetweight). At 1 h the concentrations of all three polyether analogues aresimilar in the liver (≈315 nmol/g wet weight). Interestingly, at 1 h,the concentration of the 3′-polyether (9) has significantly increasedfrom 242±38 nmol/g wet weight to 318±46 nmol/g wet weight (p<0.05),while the concentrations of 6 and 3 have decreased by 36% and 6%,respectively. At 2 and 4 h, the 3′-ligand (9) is the most concentratedin the liver, followed by the 5′-analogue (6) and 4′-analogue (3). Theliver concentration of the parent 1 is lower than the polyethers at alltime points (FIG. 4).

In the heart at 0.5 h (FIG. 4) the relative concentration of thepolyethers (6>3>9) follows the same trend as in the kidney and liver atthe same time point. However, the actual levels are much lower, <90nmol/g wet weight. The order of concentration in the heart remains thesame at 1 and 2 h. At 4 h, the 5′-ligand (6) is still the mostconcentrated chelator. Although the parent drug 1 is higher than 9 at0.5 h, it is the least concentrated ligand at all other time points(FIG. 4).

In the pancreas (FIG. 4), the relative concentration of the polyethersis 6>3>9 at all time points. The tissue content of both 3 and 9 increasefrom 0.5 to 1 h (FIG. 4). At 2 h, the levels of 3 and 6 are similar (≈30nmol/g wet weight), while the concentration of 9 is 16 nmol/g wetweight. The parent drug (1) is higher in concentration than 3 and 9 at0.5 h and similar at 1 h (FIG. 4). At 2 h, 1 is the least concentratedligand and is undetectable at 4 h.

The plasma chelator concentration data (FIG. 4) are consistent with theidea that the ligands are cleared quickly. At 0.5 h the plasma ligandlevels [6 (324±20 μM)>3 (194±60 μM)>9 (62±24 μM)] mirror what isoccurring in the liver, kidney, pancreas, and heart. At 1 h, while theorder is the same, 6 has diminished by 39%, 3 has diminished by 28%, and9 has diminished by 26%. At 2 h, 6 is now down by 54%, 3 is down by 92%,and 9 is down by 61%. At 4 h, 6 has dropped by 82%, 3 has dropped by 97%and 9 has dropped by 79%. The drop in plasma concentration of ligand 3is considerably faster than the disappearance of 6 or 9. However, 9never achieves plasma levels close to 3 and 6. The parent drug 1 is onlyhigher in concentration than 9 at 0.5 h; it is lower than all otherligands at all other time points (FIG. 4). This observation relative toliver concentrations of the chelators suggests an efficient first-passclearance of 1 and 9. Because of the excellent ICE of the 3′-polyether(9) and its moderate kidney concentrations, this ligand will be movedforward into preclinical toxicity trials. What is particularlyintriguing about this ligand is the fact that it performs so well inboth the rodents and the primates, suggesting a higher index of successin humans.

Early studies clearly demonstrated the polyether (S)-4′-(HO)-DADFT-PE(3) to be profoundly less nephrotoxic in rodents than the corresponding(S)-4′-(CH₃O)-DADFT (2) or the parent drug (S)-4′-(HO)-DADFT (1)[Bergeron, R. J., Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao,H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. The polyether 3 was also shown to have excellentiron-clearing efficiency in primates. The histopathology of kidneys ofrats treated with (S)-4′-(HO)-DADFT-PE (3) presented with significantlyfewer structural alterations in the proximal tubules than did tissuestaken from rodents exposed to the parent ligand 1 [Bergeron, R. J.,Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N. andRocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. Initial kidney tissue level measurements taken at 2 h fromanimals treated with the 4′-polyether (3) seemed consistent with thehistopathology; there was less polyether in the kidney than the parentdrug and less nephrotoxicity [Bergeron, R. J., Wiegand, J., McManis, J.S., Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. The ICE in both rodents and primates and the absence oftoxicity seen with the 4′-polyether (3) compelled the investigation ofthe impact of fixing the 3,6,9-polyether chain to the aromatic ring ofDADFT at positions other than the 4′-carbon had on ICE and ligand tissuedistribution. Two systems were chosen, (S)-3′-(HO)-DADFT-PE (9) and(S)-5′-(HO)-DADFT-PE (6) (Tables 1 and 2).

The key step in the assembly of the two ligands (Scheme 1) involvedalkylation of either 2,5-dihydroxy isopropyl ester (10) or the2,3-dihydroxy ethyl ester (11) with tri(ethylene glycol) monomethylether under Mitsunobu conditions. This alkylation was followed by esterhydrolysis. Mitsunobu alkylation was highly specific for the 5′ or the3′ and did not involve the 2′-(HO), probably for steric reasons. Theregioselectivity of the reaction was consistent with nuclear Overhausereffect difference spectra (FIGS. 1 and 2) of both polyethers 9 and 12.

While log P_(app) was a predictor of ICE in the rodents in the case ofthe methoxylated analogues versus their corresponding parents (1 vs 2),(4 vs 5), and (7 vs 8) and with the 5′-substituted ligands 4-6 inprimates, it was not a useful tool for parent versus polyether. In eachset of compounds in rodents, the ICE of the polyether was significantlygreater than that of the parent ligand (1 vs 3, 500%, p<0.003; 4 vs 6,800%, p<0.005 and 7 vs 9, 230%, p<0.05; Table 1). This suggested thatthere may well be additional parameters beyond ligand-metal access thatcontrol ICE: efficiency of the metal complex transport through variousorganic anion transports, such as cMOAT, log P_(app) of the metalcomplexes themselves, and ligand-albumin binding. The ICE differencesbetween parent and polyether, for example, (S)-4′-(HO)-DADFT (1) and(S)-4′-(HO)-DADFT-PE (3), in rodents was shown to parallelligand-albumin binding differences (Table 3). Rodents were given sodiumbenzoate, a compound known to displace ligands from Sudlow types I andII albumin binding sites, along with either 1 or 3. The scadministration of sodium benzoate increased the ICE of 1 by 10.9-fold(p<0.001). The ICE of animals given ligand 3 and sodium benzoate alsoincreased, but only by 1.6-fold (p<0.05). This may ultimately explain,at least in part, the difference in ligand ICE in primates versusrodents. The chelators may uniformly bind more weakly to primatealbumin. In the primates, the differences in ligand ICE were not asprofound and were generally within experimental error (Table 2), exceptfor 4 versus 6, in which the parent's ICE (12.6±3.0%) was greater thanthat of the corresponding polyether 6 (8.1±2.8%, p<0.05).

The effect of altering the position of the polyether on ligand-tissueconcentrations is significant (FIG. 4). The trend in all tissueconcentrations except the liver is generally (S)-5′-(HO)-DADFT-PE(6)>(S)-4′-(HO)-DADFT-PE (3)>(S)-3′-(HO)-DADFT-PE (9). In the liver at0.5 h, the concentrations are also 6>3>9>1, and at 1 h, 6≈3≈9>>1.However, beyond that time point, 9 achieves and remains at the highestconcentration. The most confounding piece of data is associated with thekidney ligand concentration of (S)-4′-(HO)-DADFT-PE (3) at time pointsearlier than 2 h. Previous studies clearly demonstrated the 4′-polyether(3) to be much less nephrotoxic than the parent drug (S)-4′-(HO)-DADFT(1) [Bergeron, R. J., Wiegand, J., McManis, J. S., Vinson, J. R. T.,Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. This was consistent with the renal tissue levels (1>3) atthe 2 h time point [Bergeron, R. J., Wiegand, J., McManis, J. S.,Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. However, in the current study 1 and 3 were found to havesimilar concentrations at 0.5 h; ligand 3 is actually slightly higherthan 1 at 1 h. Thus it seems that the reduced toxicity of the polyether3 relative to the parent 1 cannot be explained simply by kidney chelatorconcentrations.

Finally, the performance ratios (ICE_(primates)/ICE_(rodents)) ofligands 3, 6 and 9 (Table 3) are all <4.6, suggesting comparable ironclearance between the two species. Although ligand 6 has virtuallyidentical ICEs in the primates and rodents (PR=1.0), it is the leastefficient (ICE 8.1±2.8%) of the three polyethers in primates and willnot be pursued further. While 3′-(CH₃O)-DADFT (8) is the most effectivechelator in rodents (12.4±3.5%) and performs well in primates(22.5±7.1%), it is expected to have a toxicity profile (nephrotoxicity)similar to that of the 4′- and 5′-(CH₃O)-DADFT ligands 2 [Bergeron, R.J., Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N.and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783] and 5 [Bergeron, R. J., Wiegand, J., McManis, J. S. andBharti, N. The Design, Synthesis, and Evaluation of Organ-Specific IronChelators. J. Med. Chem. 2006, 49, 7032-7043], respectively, and willnot be moved forward. The (S)-3′-(HO)-DADFT-PE (9) works well in bothprimates (24.5±7.6%) and rats (10.6±4.4%), PR 2.3, suggesting a higherindex of success in a third species, humans. In addition, if therelatively large fraction of the iron excreted in the urine of themonkeys were also found in the urine of patients, performance ofiron-balance studies would be facilitated. This chelator will be movedforward into protracted preclinical toxicological assessments inrodents.

In the Examples: C. apella monkeys were obtained from World WidePrimates (Miami, Fla.). Male Sprague-Dawley rats were procured fromHarlan Sprague-Dawley (Indianapolis, Ind.). Cremophor RH-40 was acquiredfrom BASF (Parsippany, N.J.). Ultrapure salts were purchased fromJohnson Matthey Electronics (Royston, U.K.). All hematological andbiochemical studies [Bergeron, R. J., Streiff, R. R., Creary, E. A.,Daniels, R. D., Jr., King, W., Luchetta, G., Wiegand, J., Moerker, T.and Peter, H. H. A Comparative Study of the Iron-Clearing Properties ofDesferrithiocin Analogues with Desferrioxamine B in a Cebus MonkeyModel. Blood 1993, 81, 2166-2173] were performed by Antech Diagnostics(Tampa, Fla.). Atomic absorption (AA) measurements were made on aPerkin-Elmer model 5100 PC (Norwalk, Conn.). Histopathological analysiswas carried out by Florida Vet Path (Bushnell, Fla.).

Cannulation of Bile Duct in Non-Iron-Overloaded Rats has been describedpreviously. The cannulation has been described previously. Bile sampleswere collected from male Sprague-Dawley rats (400-450 g) at 3-hintervals for up to 48 h. The urine sample(s) was taken at 24 hintervals. Sample collection and handling are as previously described[Bergeron, R. J., Streiff, R. R., Creary, E. A., Daniels, R. D., Jr.,King, W., Luchetta, G., Wiegand, J., Moerker, T. and Peter, H. H. AComparative Study of the Iron-Clearing Properties of DesferrithiocinAnalogues with Desferrioxamine B in a Cebus Monkey Model. Blood 1993,81, 2166-2173; Bergeron, R. J., Streiff, R. R., Wiegand, J., Vinson, J.R. T., Luchetta, G., Evans, K. M., Peter, H. and Jenny, H.-B. AComparative Evaluation of Iron Clearance Models. Ann. N. Y. Acad. Sci.1990, 612, 378-393].

The monkeys (3.5-4 kg) were iron overloaded with intravenous irondextran as specified in earlier publications to provide about 500 mg ofiron per kg of body weight [Bergeron, R. J., Streiff, R. R., Wiegand,J., Luchetta, G., Creary, E. A. and Peter, H. H. A Comparison of theIron-Clearing Properties of 1,2-Dimethyl-3-hydroxypyrid-4-one,1,2-Diethyl-3-hydroxypyrid-4-one, and Deferoxamine. Blood 1992, 79,1882-1890]; the serum transferrin iron saturation rose to between 70 and80%. At least 20 half-lives, 60 d [Wood, J. K., Milner, P. F. andPathak, U. N. The Metabolism of Iron-dextran Given as a Total-doseInfusion to Iron Deficient Jamaican Subjects. Br. J. Haematol. 1968, 14,119-129], elapsed before any of the animals were used in experimentsevaluating iron-chelating agents.

Fecal and urine samples were collected at 24-h intervals and processedas described previously [Bergeron, R. J., Streiff, R. R., Creary, E. A.,Daniels, R. D., Jr., King, W., Luchetta, G., Wiegand, J., Moerker, T.and Peter, H. H. A Comparative Study of the Iron-Clearing Properties ofDesferrithiocin Analogues with Desferrioxamine B in a Cebus MonkeyModel. Blood 1993, 81, 2166-2173; Bergeron, R. J., Streiff, R. R.,Wiegand, J., Vinson, J. R. T., Luchetta, G., Evans, K. M., Peter, H. andJenny, H.-B. A Comparative Evaluation of Iron Clearance Models. Ann. N.Y. Acad. Sci. 1990, 612, 378-393; Bergeron, R. J., Wiegand, J. andBrittenham, G. M. HBED: A Potential Alternative to Deferoxamine forIron-Chelating Therapy. Blood 1998, 91, 1446-1452]. Briefly, thecollections began 4 d prior to the administration of the test drug andcontinued for an additional 5 d after the drug was given. Ironconcentrations were determined by flame atomic absorption spectroscopyas presented in other publications [Bergeron, R. J., Streiff, R. R.,Wiegand, J., Vinson, J. R. T., Luchetta, G., Evans, K. M., Peter, H. andJenny, H.-B. A Comparative Evaluation of Iron Clearance Models. Ann. N.Y. Acad. Sci. 1990, 612, 378-393; Bergeron, R. J., Wiegand, J.,Wollenweber, M., McManis, J. S., Algee, S. E. and Ratliff-Thompson, K.Synthesis and Biological Evaluation of Naphthyldesferrithiocin IronChelators. J. Med. Chem. 1996, 39, 1575-1581].

In the iron-clearing experiments, the rats were given a single 300μmol/kg dose of drugs 1-9 orally (po). The compounds were administeredas (1) a solution in water (3) or (2) the monosodium salt of thecompound of interest (prepared by the addition of 1 equiv of NaOH to asuspension of the free acid in distilled water (1-2, 4-9). The drugswere given to the monkeys po at a dose of 75 μmol/kg (6, 9) or 150μmol/kg (1-5, 7-8). The drugs were prepared as for the rats, except that2 and 7-8 were solubilized in 40% Cremophor RH-40/water.

The theoretical iron outputs of the chelators were generated on thebasis of a 2:1 complex. The efficiencies in the rats and monkeys werecalculated as set forth elsewhere [Bergeron, R. J., Wiegand, J.,McManis, J. S., McCosar, B. H., Weimar, W. R., Brittenham, G. M. andSmith, R. E. Effects of C-4 Stereochemistry and C-4′ Hydroxylation onthe Iron Clearing Efficiency and Toxicity of Desferrithiocin Analogues.J. Med. Chem. 1999, 42, 2432-2440]. Data are presented as the mean±thestandard error of the mean; p-values were generated via a one-tailedstudent's t-test, in which the inequality of variances was assumed; anda p-value of <0.05 was considered significant.

Collection of Tissue Distribution Samples from Rodents: MaleSprague-Dawley rats (250-350 g) were given a single sc injection of themonosodium salts of 6 and 9 prepared as described above at a dose of 300μmol/kg. At times 0.5, 1, 2, and 4 h after dosing (n=3 rats per timepoint) the animals were euthanized by exposure to CO₂ gas. Blood wasobtained via cardiac puncture into vacutainers containing sodiumcitrate. The blood was centrifuged, and the plasma was separated foranalysis. The liver, heart, kidneys, and pancreas were then removed fromthe animals. Tissue samples of animals treated with (S)-4′-(HO)-DADFT(1) and (S)-4′-(HO)-DADFT-PE (3) were prepared for HPLC analysis aspreviously described [Bergeron, R. J., Wiegand, J., McManis, J. S.,Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783]. In the current study, tissues from the (S)-3′-(HO)-DADFT-PE(9) and (S)-5′-(HO)-DADFT-PE (6) treated rats were prepared for HPLCanalysis by homogenizing them in 0.5 N HClO₄ at a ratio of 1:3 (w/v).Then, as a rinse, CH₃OH at a ratio of 1:3 (w/v) was added and themixture was stored at −20° C. for 30 min. This homogenate wascentrifuged. The supernatant was diluted with mobile phase A (95% buffer[25 mM KH₂PO₄, pH 3.0]/5% CH₃CN), vortexed, and filtered with a 0.2 μmmembrane. Analytical separation was performed on a Discovery RP AmideC₁₆ HPLC system with UV detection at 310 nm as described previously[Bergeron, R. J., Wiegand, J., Weimar, W. R., McManis, J. S., Smith, R.E. and Abboud, K. A. Iron Chelation Promoted by DesazadesferrithiocinAnalogues: An Enantioselective Barrier. Chirality 2003, 15, 593-599;Bergeron, R. J., Wiegand, J., Ratliff-Thompson, K. and Weimar, W. R. TheOrigin of the Differences in (R)- and (S)-Desmethyldesferrithiocin:Iron-Clearing Properties. Ann. N. Y. Acad. Sci. 1998, 850, 202-216].Mobile phase and chromatographic conditions were as follows: solvent A,5% CH₃CN/95% buffer; solvent B, 60% CH₃CN/40% buffer. The concentrationswere calculated from the peak area fitted to calibration curves bynonweighted least-squares linear regression with Rainin Dynamax HPLCMethod Manager software (Rainin Instrument Co.). The method had adetection limit of 0.25 μM and was reproducible and linear over a rangeof 1-1000 μM.

Tissue distribution data are presented as the mean; p-values weregenerated via a one-tailed student's t-test, in which the inequality ofvariances was assumed and a p-value of <0.05 was considered significant.

Compounds 4 and 7 were synthesized using the method published earlier[Bergeron, R. J., Wiegand, J., McManis, J. S. and Bharti, N. The Design,Synthesis, and Evaluation of Organ-Specific Iron Chelators. J. Med.Chem. 2006, 49, 7032-7043; Bergeron, R. J., Wiegand, J., McManis, J. S.,Weimar, W. R., Park, J.-H., Eiler-McManis, E., Bergeron, J. andBrittenham, G. M. Partition-Variant Desferrithiocin Analogues: OrganTargeting and Increased Iron Clearance. J. Med. Chem. 2005, 48,821-831]. Reagents were purchased from Aldrich Chemical Co. (Milwaukee,Wis.), and Fisher Optima grade solvents were routinely used. DMF wasdistilled under inert atmosphere and THF was distilled from sodium andbenzophenone. Reactions were run under a nitrogen atmosphere, andorganic extracts were dried with sodium sulfate and filtered. Silica gel40-63 from SiliCycle, Inc. was used for flash column chromatography.C-18 for reverse phase column chromatography was obtained from SigmaChemical Co. Optical rotations were run at 589 nm (sodium D line)utilizing a Perkin-Elmer 341 polarimeter, with c being the concentrationin grams of compound per 100 mL of solution in chloroform. ¹H NMRspectra were recorded at 400 MHz and chemical shifts (6) are given inparts per million downfield from tetramethylsilane for CDCl₃ (notindicated) or sodium 3-(trimethylsilyl) propionate-2, 2, 3, 3-d₄ forD₂O. ¹³C spectra were run at 100 MHz and chemical shifts (δ) are givenin parts per million referenced to the residual solvent resonance inCDCl₃ (δ 77.16). Coupling constants (J) are in hertz, and the base peaksare reported for the ESI-FTICR mass spectra. Elemental analyses wereperformed by Atlantic Microlabs (Norcross, Ga.). NOE difference spectrawere obtained at 500 MHz and samples were not degassed, were not spun,and the probe temperature was regulated at 27° C. For 12 theconcentration was 15 mg/0.6 mL in CDCl₃, and for 9 the concentration was5 mg/mL D₂O.

Separate spectra to investigate nuclear Overhauser effects (NOEs) wereacquired by low-power irradiation off-resonance and then on theresonance for the methylene hydrogens, using a 3-second presaturationperiod, a 45° pulse, and a 3-second acquisition time. Typically, 100-300acquisitions were accumulated for each pair of free induction decaysbefore processing with exponential line broadening and Fouriertransformation.

NOE difference spectra were presented by subtracting the spectrum withirradiation off-resonance from the spectrum with on-resonancepresaturation. These difference spectra were then analyzed byintegration of the relevant signals. The inverted methylene resonancesfor the two hydrogens labeled g (FIG. 1) and h (FIG. 2) were assigned anintegral value of −200%, and the integrals for the positive signalenhancements of the various other resonances were then taken as percentenhancements of their parent signals. Results are reported as theaverage enhancements from three or four replicates of each differencespectrum.

Example 1 Isopropyl2-(2,5-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylate (10)

2-Iodopropane (8.95 g, 52.65 mmol) and DIEA (6.79 g, 52.65 mmol) weresuccessively added to 4 (7.40 g, 29.25 mmol) in DMF (90 mL), and thesolution was stirred at rt for 72 h. After solvent removal under hivac,the residue was treated with 1:1, 0.5 M citric acid/saturated NaCl (300mL) and was extracted with EtOAc (250 mL, 2×100 mL). Combined organicextracts were washed with 50 mL portions of 1% NaHSO₃, H₂O, andsaturated NaCl, and the solution was evaporated. Purification by flashcolumn chromatography using 20% EtOAc in toluene generated 7.94 g of 10(92%) as a yellow oil: [α]²⁰+41.1°; ¹H NMR δ 1.27 and 1.29 (2 d, 6H,J=5.5), 1.65 (s, 3H), 3.21 (d, 1H, J=11.6), 3.85 (d, 1H, J=11.2), 5.09(septet, 1H, J=6.4), 6.89 (m, 2H,); ¹³C NMR δ 21.70, 24.36, 40.05,70.02, 83.78, 115.90, 115.94, 118.05, 121.59, 148.02, 153.11, 171.24,172.57; HRMS m/z calcd for C₁₄H₁₈NO₄S, 296.0956 (M+H). found, 296.0956.Anal. (C₁₄H₁₇NO₄S) C; H; N.

Example 2 Ethyl2-(2,3-Dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylate (11)

Iodoethane (8.61 g, 55.20 mmol) and DIEA (7.13 g, 55.20 mmol) weresuccessively added to 7 (7.36 g, 29.06 mmol) in DMF (100 mL), and thesolution was stirred at rt for 48 h. After solvent removal under hivac,the residue was treated with 1:1, 0.5 M citric acid/saturated NaCl (300mL) and was extracted with EtOAc (200 mL, 2×100 mL). Combined organiclayers were washed with 150 mL portions of 1% NaHSO₃, H₂O, and saturatedNaCl, and the solvent was evaporated. Purification by flash columnchromatography using 10% EtOAc in DCM gave 8.01 g of 11 (98%) as ayellow oil: [α]²⁰+57.41°; ¹H NMR δδ1.31 (t, 3H, J=7.2), 1.68 (s, 3H),3.25 (d, 1H, J=11.6), 3.88 (d, 1H, J=11.2), 4.26 (q, 2H, J=7.2), 5.71(br s, 1H), 6.79 (t, 1H, J=7.8), 6.97 (dd, 1H, J=8.4, 1.2), 7.03 (dd,1H, J=7.8, 1.2); ¹³C NMR δ 14.22, 24.56, 40.19, 62.17, 83.26, 115.74,117.88, 119.13, 121.20, 145.11, 146.83, 172.09, 172.69; HRMS m/z calcdfor C₁₃H₁₅NO₄SNa, 304.0619 (M+Na). found, 304.0625. Anal. (C₁₃H₁₅NO₄S)C; H; N.

Example 3 Isopropyl(S)-4,5-Dihydro-2-[2-hydroxy-5-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylate(12)

Tri(ethylene glycol) monomethyl ether (3.19 g, 19.40 mmol) anddiisopropylazodicarboxylate (4.73 g, 23.39 mmol) were successively addedto a solution of 10 (5.62 g, 19.02 mmol) and triphenylphosphine (5.93 g,22.63 mmol) in dry THF (120 mL) with ice bath cooling. The solution wasstirred at room temperature for 5 h and was maintained at 5° C. for 16h. Solvent was removed by rotary evaporation, and 40% EtOAc/petroleumether (100 mL) was added. The solution was kept at 5° C. for 12 h; thesolid formed was filtered. The filtrate was concentrated in vacuo andwas purified by column chromatography (50% EtOAc/petroleum ether) togive 4.36 g of 12 (52%) as a yellow oil: [α]²⁰+23.2°; ¹H NMR δδ 1.27 and1.29 (2d, 6H, J=6.2), 1.65 (s, 3H), 3.22 (d, 1H, J=11.2), 3.53-3.58 (m,2H), 3.64-3.71 (m, 4H), 3.72-3.77 (m, 2H), 3.84 (t, 2H, J=4.7), 3.87 (d,1H, J=11.4), 4.08-4.12 (m, 2H), 5.08 (septet, 1H, J=4.0), 6.91-6.96 (m,2H), 7.01 (dd, 1H, J=9.2, 3.2), 12.02 (br s, 1H); ¹³C NMR δ 21.71,24.37, 40.01, 59.15, 68.52, 69.69, 69.91, 70.68, 70.76, 70.92, 72.03,83.83, 115.23, 115.82, 118.03, 121.49, 151.17, 153.80, 171.12, 172.13;HRMS m/z calcd for C₂₁H₃₂NO₇S, 442.1899 (M+H). found, 442.1887. Anal.(C₂₁H₃₁NO₇S) C; H; N.

Example 4(S)-4,5-Dihydro-2-[2-hydroxy-5-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicAcid (6)

A solution of 50% (w/w) NaOH (3.34 mL, 94 mmol) in CH₃OH (34 mL) wasadded to 12 (2.14 g, 4.85 mmol) in CH₃OH (70 mL) with ice bath cooling.The reaction mixture was stirred at room temperature for 18 h, and thebulk of the solvent was removed by rotary evaporation. The residue wastreated with dilute NaCl (100 mL) and was extracted with ether (3×50mL). The basic aqueous phase was cooled in ice, acidified with 2 N HClto pH=2, and extracted with EtOAc (3×100 mL). After the EtOAc layerswere washed with saturated NaCl (100 mL), glassware that was presoakedin 3 N HCl for 15 min was employed henceforth. After solvent removal byrotary evaporation, 1.88 g of 6 (97%) was obtained as an orange oil:[α]²⁰+40.0°; ¹H NMR (D₂O) δ 1.72 (s, 3H), 3.26 (d, 1H, J=11.2), 3.38 (s,3H), 3.54-3.58 (m, 2H), 3.64-3.71 (m, 4H), 3.72-3.76 (m, 2H), 4.07-4.11(m, 3H), 6.91-6.95 (m, 2H), 7.01 (dd, 1H, J=9.0, 3.0); ¹³C NMR 624.46,40.04, 59.04, 68.40, 69.86, 70.48, 70.62, 70.80, 71.91, 83.44, 115.21,115.65, 118.09, 121.63, 151.12, 153.69, 171.83, 176.18; HRMS m/z calcdfor C₁₈H₂₆NO₇S, 400.1429 (M+H). found, 400.1416.

Example 5 Ethyl(S)-4,5-Dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylate(13)

Tri(ethylene glycol) monomethyl ether (1.70 g, 10.36 mmol) anddiisopropylazodicarboxylate (2.53 g, 12.50 mmol) were successively addedto a solution of 11 (3.0 g, 10.16 mmol) and triphenylphosphine (3.17 g,12.09 mmol) in dry THF (60 mL) with ice bath cooling. The solution wasstirred at room temperature for 8 h and was maintained at 5° C. for 40h. Solvent was removed by rotary evaporation, and 40% EtOAc/petroleumether (50 mL) was added. The solution was kept at 5° C. for 12 h; thesolid formed was filtered. The filtrate was concentrated in vacuo andwas purified by column chromatography eluting with 50% EtOAc/petroleumether to give 1.08 g of 13 (25%) as an orange oil. An analytical samplewas purified on C-18 reverse phase column eluting with equal volumes of50% aq. MeOH and 40% aq. MeOH, respectively: [α]²⁰+40.0°; ¹H NMR δδ1.30(t, 3H, J=7.2), 1.66 (s, 3H), 3.23 (d, 1H, J=11.2), 3.38 (s, 3H),3.52-3.58 (m, 2H), 3.63-3.71 (m, 4H), 3.74-3.79 (m, 2H), 3.88 (d, 1H,J=11.6), 3.91 (t, 2H, J=5.0), 4.20-4.26 (m, 4H), 6.79 (t, 1H, J=7.6),7.01-7.07 (m, 2H); ¹³C NMR δ 14.18, 24.47, 39.96, 59.10, 62.07, 68.95,69.79, 70.60, 70.70, 70.91, 71.99, 83.48, 116.42, 117.62, 118.29,122.71, 147.72, 150.35, 171.70, 172.69; HRMS m/z calcd for C₂₀H₂₉NO₇SNa,450.1562 (M+Na). found, 450.1568. Anal. (C₂₀H₂₉NO₇S) C; H; N.

Example 6(S)-4,5-Dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicAcid (9)

A solution of 50% (w/w) NaOH (13.88 mL, 266.02 mmol) in CH₃OH (120 mL)was added to 13 (8.89 g, 20.80 mmol) in CH₃OH (280 mL) with ice bathcooling. The reaction mixture was stirred at room temperature for 6 h,and the bulk of the solvent was removed by rotary evaporation. Theresidue was treated with dilute NaCl (300 mL) and the basic aqueousphase was cooled in ice, acidified with 2 N HCl to pH=2, and extractedwith EtOAc (4×150 mL). After the EtOAc layers were washed with saturatedNaCl (300 mL), glassware that was presoaked in 3 N HCl for 15 min wasemployed henceforth. After solvent removal by rotary evaporation,purification was done on C-18 reverse phase column, eluting with 50% aq.methanol and lyophilized to furnish 4.98 g of 9 (60%) as an orange oil:[α]²⁰+61.9°; ¹H NMR (D₂O) δ 1.77 (s, 3H), 3.35 (s, 3H), 3.56-3.62 (m,3H), 3.64-3.73 (m, 4H), 3.75-3.89 (m, 2H), 3.92-3.96 (m, 2H), 3.99 (d,1H, J=11.6), 4.25-4.31 (m, 2H), 6.99 (t, 1H, J=8.2), 7.26-7.33 (m, 2H);¹³C NMR δ 24.52, 39.93, 59.07, 69.04, 69.83, 70.49, 70.64, 70.86, 71.97,83.21, 116.33, 117.94, 118.50, 122.80, 147.67, 150.24, 172.38, 176.10;HRMS m/z calcd for C₁₈H₂₆NO₇S, 400.1429 (M+H). found, 400.1413.

TABLE 1 Iron-Clearing Activity of Desferrithiocin Analogues WhenAdministered Orally to Rodents and the Partition Coefficients of theCompounds Iron- Clearing Efficiency Desferrithiocin Analogue (%)^(a) logP_(app) ^(b)

 1.1 ± 0.8^(c) [100/0] −1.05

 6.6 ± 2.8^(c)  [98/2] −0.70

 5.5 ± 1.9^(c)  [90/10] −1.10

 1.0 ± 0.9^(d)  [99/1] −1.14

 6.3 ± 1.2^(d)  [95/5] −0.61

 8.0 ± 1.8  [98/2] −1.27

 4.6 ± 0.9  [98/2] −1.17

12.4 ± 3.5^(e)  [99/1] −1.12

10.6 ± 4.4^(e)  [95/5] −1.22

a. In the rodents [n=3 (6), 4 (2, 4, 5, 7, 9), 5 (3, 8), or 8 (1)], thedose was 300 μmol/kg. The compounds were solubilized in either distilledwater (3) or were given as their monosodium salts, prepared by theaddition of 1 equiv of NaOH to a suspension of the free acid indistilled water (1, 2, 4-9). The efficiency of each compound wascalculated by subtracting the iron excretion of control animals from theiron excretion of the treated animals. This number was then divided bythe theoretical output; the result is expressed as a percent. Therelative percentages of the iron excreted in the bile and urine are inbrackets.

b. Data are expressed as the log of the fraction in the octanol layer(log P_(app)); measurements were done in TRIS buffer, pH 7.4, using a“shake flask” direct method. The values obtained for compounds 1 and 2are from Bergeron et al, J. Med. Chem. 2003, 46, 1470-1477; the valuefor 3 is from Bergeron et al, J. Med. Chem. 2006, 49, 2772-2783; thevalues for 4 and 5 are from Bergeron et al, J. Med. Chem. 2006, 49,7032-7043; and the values for 7 and 8 are from Bergeron et al, J. Med.Chem. 2005, 48, 821-831.

c. Data are from ref Bergeron et al, J. Med. Chem. 2006, 49, 2772-2783.

d. Data are from ref Bergeron et al, J. Med. Chem. 2006, 49, 7032-7043.

e. ICE is based on a 48 h sample collection period.

TABLE 2 Iron-Clearing Activity of Desferrithiocin Analogues WhenAdministered Orally to Cebus apella Primates and the PartitionCoefficients of the Compounds Iron- Clearing Efficiency DesferrithiocinAnalogue (%)^(a) log P_(app) ^(b)

16.8 ± 7.2^(c) [88/12] −1.05

24.4 ± 10.8^(d) [91/9] −0.70

25.4 ± 7.4^(e) [96/4] −1.10

12.6 ± 3.0^(f) [88/12] −1.14

18.9 ± 2.3^(f) [94/6] −0.61

 8.1 ± 2.8^(g) [56/44] −1.27

23.1 ± 5.9^(h) [83/17] −1.17

22.5 ± 7.1^(h) [91/9] −1.12

24.5 ± 7.6^(g) [72/28] −1.22

a. In the monkeys [n=4 (3-7), 5 (8), 6 (1), or 7 (2, 9)]. The drugs weregiven po at a dose of 75 μmol/kg (6, 9) or 150 μmol/kg (1-5, 7-8). Thecompounds were solubilized in either distilled water (3), 40% Cremophor(2, 7, 8), or were given as their monosodium salts, prepared by theaddition of 1 equiv of NaOH to a suspension of the free acid indistilled water (1, 4-6, 9). The efficiency of each compound wascalculated by averaging the iron output for 4 days before theadministration of the drug, subtracting these numbers from the two-dayiron clearance after the administration of the drug, and then dividingby the theoretical output; the result is expressed as a percent. Therelative percentages of the iron excreted in the stool and urine are inbrackets.

b. Data are expressed as the log of the fraction in the octanol layer(log P_(app)); measurements were done in TRIS buffer, pH 7.4, using a“shake flask” direct method. The values obtained for compounds 1 and 2are from ref Bergeron et al, J. Med. Chem. 2003, 46, 1470-1477; thevalue for 3 is from ref Bergeron et al, J. Med. Chem. 2006, 49,2772-2783; the values for 4 and 5 are from ref [Bergeron, R. J.,Wiegand, J., McManis, J. S. and Bharti, N. The Design, Synthesis, andEvaluation of Organ-Specific Iron Chelators. J. Med. Chem. 2006, 49,7032-7043]; the values for 7 and 8 are from Bergeron et al, J. Med.Chem. 2005, 48, 821-831.

c. Data are from Bergeron et al, J. Med. Chem. 1999, 42, 2432-2440.

d. Data are from Bergeron et al, J. Med. Chem. 2003, 46, 1470-1477.

e. Data are from Bergeron et al, J. Med. Chem. 2006, 49, 2772-2783.

f. Data are from Bergeron et al, J. Med. Chem. 2006, 49, 7032-7043.

g. The dose was 75 μmol/kg.

h. Data are from Bergeron et al, J. Med. Chem. 2005, 48, 821-831.

TABLE 3 Iron-Clearing Efficiency Performance Ratios of DesferrithiocinAnalogues in Primates versus Rodents ICE Primate/ DesferrithiocinAnalogue ICE Rodent

15.3 (S)-4′-(HO)-DADFT, 1

3.7 (S)-4′-(CH₃O)-DADFT, 2

4.6 (S)-4′-(HO)-DADFT-PE, 3

12.6 (S)-5′-(HO)-DADFT, 4

3.0 (S)-5′-(CH₃O)-DADFT, 5

1.0 (S)-5′-(HO)-DADFT-PE, 6

5.0 (S)-3′-(HO)-DADFT, 7

1.8 (S)-3′-(CH₃O)-DADFT, 8

2.3 (S)-3′-(HO)-DADFT-PE, 9

TABLE 4 Ligand-Albumin Binding in Rodents treated with SodiumBenzoate^(a) iron-clearing experiment dose route N efficiency (%) sodiumbenzoate 250 mg/kg/ sc 5 baseline iron dose excretion (S)-4′-(HO)-DADFT300 μmol/kg po 8 1.1 ± 0.8 (1) (S)-4′-(HO)-DADFT 300 μmol/kg po and 512.0 ± 2.6^(b) (1) plus sodium benzoate and 250 mg/ sc, re- kg/dose,spectively respectively (S)-4′-(HO)-DADFT-PE 300 μmol/kg po 5 5.5 ± 1.9(3) (S)-4′-(HO)-DADFT-PE 300 μmol/kg po and 4  8.8 ± 2.4^(c) (3) plussodium benzoate and 250 mg/ sc, re- kg/dose, spectively respectively

a. Ligand 1 was administered po as its monosodium salt, prepared by theaddition of 1 equiv of NaOH to a suspension of the free acid indistilled water. Ligand 3 was dissolved in distilled water and given po.Sodium benzoate was dissolved in distilled water and given sc at 250mg/kg/dose×6 doses. The first dose of sodium benzoate was given 0.5 hprior to the chelators; additional doses were given hourly thereafterfor the next 5 h.

b. p<0.001 vs non-benzoate 1 treated animals.

c. p<0.05 vs non-benzoate 3 treated animals.

The invention also includes enantiomers and mixtures of enantiomers(e.g., racemic mixtures) of the compounds represented by the aboveformulas along with their salts (e.g., pharmaceutically acceptablesalts), solvates and hydrates. Compounds of the invention can exist inoptically active forms that have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L or R and S are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand 1 or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or I meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these compounds, calledstereoisomers, are identical except that one or more chiral carbons arenon-superimposable mirror images of one another. A specificstereoisomer, which is an exact minor image of another stereoisomer, canalso be referred to as an enantiomer, and a mixture of such isomers isoften called an enantiomeric mixture. A 50:50 mixture of enantiomers isreferred to as a racemic mixture.

Many of the compounds described herein can have one or more chiralcenters and therefore can exist in different enantiomeric forms. Ifdesired, a chiral carbon can be designated with an asterisk (*). In thepresent application, the chiral carbon at the 4-position of thethiazoline or thiazolidine ring can be designated with an asterisk,because the configuration of this carbon is of particular interest. Whenbonds to chiral carbons are depicted as straight lines in the formulasof the invention, it is understood that both the (R) and (S)configurations of each chiral carbon, and hence both enantiomers andmixtures thereof, are embraced within the formula. As is used in theart, when it is desired to specify the absolute configuration about achiral carbon, a bond to the chiral carbon can be depicted as a wedge(bonds to atoms above the plane) and another can be depicted as a seriesor wedge of short parallel lines (bonds to atoms below the plane). TheCahn-Ingold-Prelog system can be used to assign the (R) or (S)configuration to a chiral carbon. A chiral carbon at the 4-position of athiazoline or thiazolidine ring preferably has an (S) configuration.

When compounds of the present invention contain one chiral center,compounds not prepared by an asymmetric synthesis exist in twoenantiomeric forms and the present invention includes either or bothenantiomers and mixtures of enantiomers, such as the specific 50:50mixture referred to as a racemic mixture. The enantiomers can beresolved by methods known to those skilled in the art, for example, byformation of diastereoisomeric salts that may be separated, for example,by crystallization (See, CRC Handbook of Optical Resolutions viaDiastereomeric Salt Formation by David Kozma (CRC Press, 2001));formation of diastereoisomeric derivatives or complexes that may beseparated, for example, by crystallization, gas-liquid or liquidchromatography; selective reaction of one enantiomer with anenantiomer-specific reagent, for example, enzymatic esterification; orgas-liquid or liquid chromatography in a chiral environment, forexample, on a chiral support (e.g., silica with a bound chiral ligand)or in the presence of a chiral solvent. It will be appreciated thatwhere the desired enantiomer is converted into another chemical entityby one of the separation procedures described above, a further step isrequired to liberate the desired enantiomeric form.

Alternatively, specific enantiomers may be synthesized by asymmetricsynthesis using optically active reagents, substrates, catalysts orsolvents, or by converting one enantiomer into the other by asymmetrictransformation.

Designation of a specific absolute configuration at a chiral carbon ofthe compounds of the invention is understood to mean that the designatedenantiomeric form of the compounds is in enantiomeric excess (ee) or, inother words, is substantially free from the other enantiomer. Forexample, the “R” forms of the compounds are substantially free from the“S” forms of the compounds and are, thus, in enantiomeric excess of the“S” forms. Conversely, “S” forms of the compounds are substantially freeof “R” forms of the compounds and are, thus, in enantiomeric excess ofthe “R” forms. Enantiomeric excess, as used herein, is the presence of aparticular enantiomer at greater than 50% in an enantiomeric mixture.For example, when a mixture contains 80% of a first enantiomer and 20%of a second enantiomer, the enantiomeric excess of the first enantiomeris 60%. In the present invention, the enantiomeric excess can be about20% or more, particularly about 40% or more, more particularly about 60%or more, such as about 70% or more, for example about 80% or more, suchas about 90% or more. In a particular embodiment when a specificabsolute configuration is designated, the enantiomeric excess ofdepicted compounds is at least about 90%. In a more particularembodiment, the enantiomeric excess of the compounds is at least about95%, such as at least about 97.5%, for example, at least about 99%enantiomeric excess. When a compound of the present invention has two ormore chiral carbons where R4 and R5 are not the same, it can have morethan two optical isomers and can exist in diastereomeric forms. Forexample, when there are two chiral carbons, the compound can have up to4 optical isomers and 2 pairs of enantiomers ((S,S)/(R,R) and(R,S)/(S,R>>. The pairs of enantiomers (e.g., (S,S)/(R,R>> are mirrorimage stereoisomers of one another. The stereo isomers which are notminor-images (e.g., (S,S) and (R,S>> are diastereomers. Thediastereomeric pairs may be separated by methods known to those skilledin the art, for example, chromatography or crystallization and theindividual enantiomers within each pair may be separated as describedabove. The present invention includes each diastereomer of suchcompounds and mixtures thereof.

An alkyl group is a saturated hydrocarbon in a molecule that is bondedto one other group in the molecule through a single covalent bond fromone of its carbon atoms. Alkyl groups can be cyclic or acyclic, branchedor unbranched (straight chained). An alkyl group typically has from 1 toabout 14 carbon atoms, for example, one to about six carbon atoms or oneto about four carbon atoms. Lower alkyl groups have one to four carbonatoms and include methyl, ethyl, n-propyl, iso-propyl, n-butyl,see-butyl and tert-butyl.

When cyclic, an alkyl group typically contains from about 3 to about 10carbons, for example, from about 3 to about 8 carbon atoms, e.g., acyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexylgroup, a cycloheptyl group or a cyclooctyl group.

Aryl groups include carbocyclic aromatic groups such as phenyl, p-tolyl,1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl. Aryl groups alsoinclude heteroaromatic groups such as N-imidazolyl, 2-imidazolyl,2-thienyl, 3-thienyl, 2furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl,4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl, 3-pyrazolyl,4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl, 4-thiazolyl,5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl.

Aryl groups also include fused polycyclic aromatic ring systems in whicha carbocyclic, alicyclic, or aromatic ring or heteroaryl ring is fusedto one or more other heteroaryl or aryl rings. Examples include2-benzothienyl, 3-benzothienyl, 2benzofuranyl, 3-benzofuranyl,2-indolyl, 3-indolyl, 2-quinolinyl, 3-quinolinyl, 2-benzothiazolyl,2-benzoxazolyl, 2-benzimidazolyl, 1-isoquinolinyl, 3-isoquinolinyl,1-isoindolyl and 3-isoindolyl.

Also included in the present invention are salts and pharmaceuticallyacceptable salts of the compounds described herein. Compounds disclosedherein that possess a sufficiently acidic functional group, asufficiently basic functional group or both, can react with a number oforganic or inorganic bases, and inorganic and organic acids, to formsalts. Acidic groups can form salts with one or more of the metalslisted above, along with alkali and alkaline earth metals (e.g., sodium,potassium, magnesium, calcium). In addition, acidic groups can formsalts with amines. Compounds of the invention can be supplied as atransition, lanthanide, actinide or main group metal salt. For example,the salt can be a Ga (III) salt of a compound. As a transition,lanthanide, actinide or main group metal salt, compounds of theinvention tend to form a complex with the metal. For example, if acompound of the invention is tridentate and the metal it forms a saltwith has six coordinate sites, then a 2 to 1 compound to metal complexis formed. The ratio of compound to metal will vary according to thedenticity of the metal and the number of coordination sites on the metal(preferably each coordination site is filled by a compound of theinvention, although a coordination site can be filled with other anionssuch as hydroxide, halide or a carboxylate).

Alternatively, the compound can be a substantially metal-free (e.g.iron-free) salt. Metal-free salts are not typically intended toencompass alkali and alkali earth metal salts. Metal-free salts areadvantageously administered to a subject suffering from, for example, ametal overload condition or to an individual suffering from toxic metalexposure or from focal concentrations of metals causing untowardeffects.

Acids commonly employed to form acid addition salts from compounds withbasic groups are inorganic acids such as hydrochloric acid, hydrobromicacid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, andorganic acids such as p-toluenesulfonic acid, methanesulfonic acid,oxalic acid, p-bromophenyl-sulfonic acid, carbonic acid, succinic acid,citric acid, benzoic acid, acetic acid, and the like. Examples of suchsalts include the hydroxide, sulfate, pyrosulfate, bisulfate, sulfite,bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate,metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate,propionate, decanoate, caprylate, acrylate, formate, isobutyrate,caproate, heptanoate, propiolate, oxalate, malonate, succinate,suberate, sebacate, fumarate, maleate, butyne-1,4-dioate,hexyne-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate,xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate,citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate,methanesulfonate, propanesulfonate, naphthalene-I-sulfonate,naphthalene-2sulfonate, mandelate, and the like.

The compounds disclosed herein can be prepared in the form of theirhydrates, such as hemihydrate, monohydrate, dihydrate, trihydrate,tetrahydrate and the like and as solvates.

Subjects suffering from a pathological condition responsive to chelationor sequestration of a trivalent metal can be treated with atherapeutically or prophylactically effective amount of a compound orpharmaceutical compound of the invention. One particular type ofpathological condition that is responsive to chelation of a trivalentmetal is a trivalent metal overload condition (e.g., an iron overloadcondition, an aluminum overload condition, a chromium overloadcondition). Another type of pathological condition that is responsive tometal chelation or sequestration is when the amount of free trivalentmetal is elevated (e.g., in the serum or in a cell), such as when thereis insufficient storage capacity for trivalent metals or an abnormalityin the metal storage system that leads to metal release.

Iron overload conditions or diseases can be characterized by global ironoverload or focal iron overload. Global iron overload conditionsgenerally involve an excess of iron in multiple tissues or excess ironlocated throughout an organism. Global iron overload conditions canresult from excess uptake of iron by a subject, excess storage and/orretention of iron, from, for example, dietary iron or bloodtransfusions. One global iron overload condition is primaryhemochromatosis, which is typically a genetic disorder. A second globaliron overload condition is secondary hemochromatosis, which is typicallythe result of receiving multiple (chronic) blood transfusions. Bloodtransfusions are often required for subjects suffering from thalassemiaor sickle cell anemia. A type of dietary iron overload is referred to asBantu siderosis, which is associated with the ingestion of home-brewedbeer with high iron content.

In focal iron overload conditions, the excess iron is limited to one ora few cell types or tissues or a particular organ. Alternatively,symptoms associated with the excess iron are limited to a discreteorgan, such as the heart, lungs, liver, pancreas, kidneys or brain. Itis believed that focal iron overload can lead to neurological orneurodegenerative disorders such as Parkinson's disease, Alzheimer'sdisease, Huntington's disease, neuroferritinopathy, amyotrophic lateralsclerosis and multiple sclerosis. Pathological conditions that benefitfrom metal chelation or sequestration are often associated withdeposition of the metal in the tissues of a subject. Deposition canoccur globally or focally, as described above.

A subject in need of oxidative stress reduction can have one or more ofthe following conditions: decreased levels of reducing agents, increasedlevels of reactive oxygen species, mutations in or decreased levels ofantioxidant enzymes (e.g., Cu/Zn superoxide dismutase, Mn superoxidedismutase, glutathione reductase, glutathione peroxidase, thioredoxin,thioredoxin peroxidase, DT-diaphorase), mutations in or decreased levelsof metal-binding proteins (e.g., transferrin, ferritin, ceruloplasmin,albumin, metallothionein), mutated or overactive enzymes capable ofproducing superoxide (e.g., nitric oxide synthase, NADPH oxidases,xanthine oxidase, NADH oxidase, aldehyde oxidase, dihydroorotatedehydrogenase, cytochrome c oxidase), and radiation injury. Increased ordecreased levels of reducing agents, reactive oxygen species, andproteins are determined relative to the amount of such substancestypically found in healthy persons.

A subject in need of oxidation stress reduction can be suffering from anischemic episode. Ischemic episodes can occur when there is mechanicalobstruction of the blood supply, such as from arterial narrowing ordisruption. Myocardial ischemia, which can give rise to angina pectorisand myocardial infarctions, results from inadequate circulation of bloodto the myocardium, usually due to coronary artery disease. Ischemicepisodes in the brain that resolve within 24 hours are referred to astransient ischemic attacks. A longer-lasting ischemic episode, a stroke,involves irreversible brain damage, where the type and severity ofsymptoms depend on the location and extent of brain tissue whose accessto blood circulation has been compromised. A subject at risk ofsuffering from an ischemic episode typically suffers fromatherosclerosis, other disorders of the blood vessels, increasedtendency of blood to clot, or heart disease. The compounds of thisinvention can be used to treat these disorders.

A subject in need of oxidation stress reduction can be suffering frominflammation. Inflammation is a fundamental pathologic processconsisting of a complex of cytologic and chemical reactions that occurin blood vessels and adjacent tissues in response to an injury orabnormal stimulation caused by a physical, chemical, or biologic agent.Inflammatory disorders are characterized inflammation that lasts for anextended period (i.e., chronic inflammation) or that damages tissue.Such inflammatory disorders can affect a wide variety of tissues, suchas respiratory tract, joints, bowels, and soft tissue. The compounds ofthis invention can be used to treat these disorders.

Although not bound by theory, it is believed that the compounds of theinvention derive their ability to reduce oxidative stress throughvarious mechanisms. In one mechanism, the compound binds to a metal,particularly a redox-active metal (e.g., iron), and fills all of thecoordination sites of the metal. When all of the metal coordinationsites are filled, it is believed that oxidation and/or reducing agentshave a diminished ability to interact with the metal and cause redoxcycling. In another mechanism, the compound stabilizes the metal in aparticular oxidation state, such that it is less likely to undergo redoxcycling. In yet another mechanism, the compound itself has antioxidantactivity (e.g., free radical scavenging, scavenging of reactive oxygenor nitrogen species). Desferrithiocin and its derivatives and analoguesare known to have intrinsic antioxidant activity, as described in U.S.Application Publication No. 2004/0044220, published Mar. 4, 2004, andU.S. Application Publication No. 2004/0132789, published Jul. 8, 2004and PCT Application No. WO2004/017959, published Mar. 4, 2004, USApplication Publication No. 2003/0236417, published Dec. 25, 2003, andU.S. Pat. Nos. 6,083,966, 6,559,315, 6,525,080, 6,521,652 the contentsof each of which are incorporated herein by reference.

It has also been reported that the reduction of iron overloads in humanscan aid in the prevention of or control of the growth of cancer [Ozaki,et al, JAMA, Feb. 14, 2007-Vol. 297, No. 6, pp 603-610; Kalinowski etal, “The evolution of iron chelators for the treatment of iron overloaddisease and cancer”, Pharmacological Reviews, vol 57, 4 pgs 547-83(2005); Taetle et al, “Combination Iron Depletion Therapy”, J. Nat.Cancer Inst., 81, 1229-1235 (1989); Bergeron et al, “Influence of Ironon in vivo Proliferation and Lethality of L1210 Cells”, J. Nutr., 115,369-374 (1985)]. Indeed, Ozaki et al report that the hypothesis thataccumulated iron contributes to disease risk through iron-catalyzed freeradical-mediated damage to critical biomolecules and through alteredcellular function rests on secure biochemical grounds. However, theyalso report that the relationship between iron and disease has remainedessentially hidden because of inconsistent findings. Finally Osaki et alreport data that support findings of a pronounced “all-cause mortalitydecrease” associated with iron levels reduced to normal levels inhumans.

A “subject” is typically a human, but can also be an animal in need oftreatment, e.g., companion animals (e.g., dogs, cats, and the like),farm animals (e.g., cows, pigs, horses, sheep, goats and the like) andlaboratory animals (e.g., rats, mice, guinea pigs, non-human primatesand the like).

The compounds and pharmaceutical compositions of the present inventioncan be administered by an appropriate route. Suitable routes ofadministration include, but are not limited to, orally,intraperitoneally, subcutaneously, intramuscularly, transdermally,rectally, sub lingually, intravenously, buccally or via inhalation.Preferably, compounds and pharmaceutical compositions of the inventionare administered orally.

The pharmaceutical compositions of the invention preferably contain apharmaceutically acceptable carrier or diluent suitable for renderingthe compound or mixture administrable orally, parenterally,intravenously, intradermally, intramuscularly or subcutaneously,rectally, via inhalation or via buccal administration, or transdermally.

The active ingredients may be admixed or compounded with a conventional,pharmaceutically acceptable carrier or diluent. It will be understood bythose skilled in the art that a mode of administration, vehicle orcarrier conventionally employed and which is inert with respect to theactive agent may be utilized for preparing and administering thepharmaceutical compositions of the present invention. Illustrative ofsuch methods, vehicles and carriers are those described, for example, inRemington's Pharmaceutical Sciences, 18th ed. (1990), the disclosure ofwhich is incorporated herein by reference.

The formulations of the present invention for use in a subject comprisethe agent, together with one or more acceptable carriers or diluentstherefore and optionally other therapeutic ingredients. The carriers ordiluents must be “acceptable” in the sense of being compatible with theother ingredients of the formulation and not deleterious to therecipient thereof. The formulations can conveniently be presented inunit dosage form and can be prepared by any of the methods well known inthe art of pharmacy. All methods include the step of bringing intoassociation the agent with the carrier or diluent which constitutes oneor more accessory ingredients. In general, the formulations are preparedby uniformly and intimately bringing into association the agent with thecarriers and then, if necessary, dividing the product into unit dosagesthereof.

Forms suitable for oral administration include tablets, troches,capsules, elixirs, suspensions, syrups, wafers, chewing gum or the likeprepared by art recognized procedures. The amount of active compound insuch therapeutically useful compositions or preparations is such that asuitable dosage will be obtained.

A syrup formulation will generally consist of a suspension or solutionof the compound or salt in a liquid carrier, for example, ethanol,glycerine or water, with a flavoring or coloring agent. Where thecomposition is in the form of a tablet, one or more pharmaceuticalcarriers routinely used for preparing solid formulations can beemployed. Examples of such carriers include magnesium stearate, starch,lactose and sucrose. Where the composition is in the form of a capsule,the use of routine encapsulation is generally suitable, for example,using the aforementioned carriers in a hard gelatin capsule shell. Wherethe composition is in the form of a soft gelatin shell capsule,pharmaceutical carriers routinely used for preparing dispersions orsuspensions can be considered, for example, aqueous gums, celluloses,silicates or oils, and are incorporated in a soft gelatin capsule shell.

Formulations suitable for parenteral administration conveniently includesterile aqueous preparations of the agents that are preferably isotonicwith the blood of the recipient. Suitable carrier solutions includephosphate buffered saline, saline, water, lactated ringers or dextrose(5% in water). Such formulations can be conveniently prepared byadmixing the agent with water to produce a solution or suspension, whichis filled into a sterile container and sealed against bacterialcontamination. Preferably, sterile materials are used under asepticmanufacturing conditions to avoid the need for terminal sterilization.

Such formulations can optionally contain one or more additionalingredients, which can include preservatives such as methylhydroxybenzoate, chlorocresol, metacresol, phenol and benzalkoniumchloride. Such materials are of special value when the formulations arepresented in multidose containers.

Buffers can also be included to provide a suitable pH value for theformulation.

Suitable buffer materials include sodium phosphate and acetate. Sodiumchloride or glycerin can be used to render a formulation isotonic withthe blood.

If desired, a formulation can be filled into containers under an inertatmosphere such as nitrogen and can be conveniently presented in unitdose or multi-dose form, for example, in a sealed ampoule.

Those skilled in the art will be aware that the amounts of the variouscomponents of the compositions of the invention to be administered inaccordance with the method of the invention to a subject will dependupon those factors noted above.

A typical suppository formulation includes the compound or apharmaceutically acceptable salt thereof which is active whenadministered in this way, with a binding and/or lubricating agent, forexample, polymeric glycols, gelatins, cocoa-butter or other low meltingvegetable waxes or fats.

Typical transdermal formulations include a conventional aqueous ornon-aqueous vehicle, for example, a cream, ointment, lotion or paste orare in the form of a medicated plastic, patch or membrane.

Typical compositions for inhalation are in the form of a solution,suspension or emulsion that can be administered in the form of anaerosol using a conventional propellant such as dichlorodifluoromethaneor trichlorofluoromethane.

The therapeutically effective amount of a compound or pharmaceuticalcomposition of the invention depends, in each case, upon severalfactors, e.g., the health, age, gender, size and condition of thesubject to be treated, the intended mode of administration, and thecapacity of the subject to incorporate the intended dosage form, amongothers. A therapeutically effective amount of an active agent is anamount sufficient to have the desired effect for the condition beingtreated. In a method of treating a subject with a condition treatable bychelating or sequestering a metal ion, a therapeutically effectiveamount of an active agent is, for example, an amount sufficient toreduce the burden of the metal in the subject, reduce the symptomsassociated with the metal ion or prevent, inhibit or delay the onsetand/or severity of symptoms associated with the presence of the metal.In a method of reducing oxidative stress in a subject in need oftreatment thereof, a therapeutically effective amount of an active agentis, for example, an amount sufficient to reduce symptoms associated withoxidative stress or prevent, inhibit or delay the onset and/or severityof symptoms associated with oxidative stress. A typical total daily doseof a compound of the invention to be administered to a subject (assumingan average 70 kg subject) is from approximately 10 mg to 1.0 g.

An alternative approach to the syntheses of both(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-4′-(HO)-DADFT-PE, 3] and[(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-3′-(HO)-DADFT-PE, 9] is described below.

In the method described elsewhere [Bergeron, R. J., Wiegand, J.,McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N. and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783], the synthesis of(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-4′-(HO)-DADFT-PE, 3] is carried out: (S)-4′-(HO)-DADFT (1) wasconverted to its isopropyl ester in 99% yield. Alkylation of the4′-hydroxyl using tri(ethylene glycol) monomethyl ether under Mitsunobuconditions (diisopropyl azodicarboxylate and triphenylphosphine in THF),filtration, and chromatography gave the isopropyl ester of 3 in 76%yield. Saponification of the ester furnished (S)-4′-(HO)-DADFT-PE (3) in95% yield, providing an overall yield of 71%.

Alternatively, selective alkylation of the ethyl ester of(S)-4′-(HO)-DADFT (14) is accomplished by heating the tosylate oftri(ethylene glycol) monomethyl ether (15, 1.0 equiv) and potassiumcarbonate (2.0 equiv) in acetone, providing masked chelator 16 in 82%yield (FIG. 6; Scheme 1). Cleavage of ethyl ester 16 as before afforded(S)-4′-(HO)-DADFT-PE (3) in 95% yield. The new route to ligand 3proceeds in greater overall yield, 78% vs. 71%; moreover, attachment ofthe polyether chain in Scheme 1 employs inexpensive reagents withoutformation of triphenylphosphine oxide and diisopropyl1,2-hydrazinedicarboxylate, simplifying purification.

In the synthesis described above, the synthesis of(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-3′-(HO)-DADFT-PE, 9] is carried out:(S)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylicacid, which was made in 88% yield from amino acid cyclization with theappropriate nitrile, was converted to its ethyl ester in 98% yield.However, the two remaining steps to chelator 9 proceeded in only 15%yield. The polyether chain was appended to the 3′-hydroxyl underMitsunobu conditions, producing the ethyl ester of 9 in 25% yield. Esterhydrolysis furnished 9 in 60% yield after purification on a reversephase column, providing an overall yield of 13%.

A more efficient syntheses of (S)-3′-(HO)-DADFT-PE (9) is presented inFIG. 6; Scheme 2. The less hindered phenolic group of2,3-dihydroxybenzonitrile (17) [Bergeron, R. J., Wiegand, J., McManis,J. S., Weimar, W. R., Park, J.-H., Eiler-McManis, E., Bergeron, J. andBrittenham, G. M. Partition-Variant Desferrithiocin Analogues: OrganTargeting and Increased Iron Clearance. J. Med. Chem. 2005, 48, 821-831]was alkylated with tosylate 15 (1.3 equiv) and sodium hydride (2.1equiv) in DMSO at room temperature, generating 18 in 70% chromatographedyield. Thus the triether chain has been attached in nearly three timesthe yield compared to the Mitsunobu coupling while avoiding thetroublesome by-products. Cyclocondensation of nitrile 18 with(S)-alpha-methyl cysteine (19) in aqueous CH₃OH buffered at pH 6completed the synthesis of (S)-3′-(HO)-DADFT-PE (9) in 90% yield. Sincethe unusual amino acid 19 was not introduced until the last step ofScheme 2, the carboxyl group did not require protection. The overallyield to 9 is 63%, much higher than from the previous route.

Example 7

(S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicAcid (3). A solution of 50% (w/w) NaOH (10.41 mL, 199.5 mmol) in CH₃OH(90 mL) was added to 16 (6.54 g, 15.3 mmol) in CH₃OH (200 mL) with icebath cooling. The reaction mixture was stirred at room temperature for16 h, and the bulk of the solvent was removed by rotary evaporation. Theresidue was treated with dilute NaCl (150 mL) and was extracted withether (3×150 mL). The basic aqueous phase was cooled in ice, acidifiedwith 2 N HCl to a pH≈2, and extracted with EtOAc (4×100 mL). The EtOAcextracts were washed with saturated NaCl (200 mL) and were concentratedin vacuo. Drying under high vacuum furnished 5.67 g of 3[Bergeron, R.J., Wiegand, J., McManis, J. S., Vinson, J. R. T., Yao, H., Bharti, N.and Rocca, J. R.(S)-4,5-Dihydro-2-(2-hydroxy-4-hydroxyphenyl)-4-methyl-4-thiazolecarboxylicAcid Polyethers: A Solution to Nephrotoxicity. J. Med. Chem. 2006, 49,2772-2783] (92%) as an orange oil: [α]²⁵+53.1° (c 0.98); ¹H NMR (D₂O) δ1.76 (s, 3H), 3.35 (s, 3H), 3.54-3.61 (m, 3H), 3.64-3.72 (m, 4H),3.74-3.78 (m, 2H), 3.90-3.94 (m, 2H), 3.96 (d, 1H, J=12.0), 4.25-4.29(m, 2H), 6.53 (d, 1H, J=2.4), 6.64 (dd, 1H, J=9.0, 2.2), 7.61 (d, 1H,J=9.2); ¹³C NMR (D₂O) δ 23.65, 39.56, 58.65, 68.34, 69.33, 70.07, 70.18,70.44, 71.62, 77.58, 102.11, 106.72, 109.66, 134.67, 161.27, 167.07,176.86, 180.70. Anal. (C₁₈H₂₅NO₇S) C; H; N.

Example 8

(S)-4,5-Dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicAcid (9). Compound 18 (7.63 g, 27.1 mmol), degassed 0.1 M pH 5.95phosphate buffer (200 mL), 19 (6.98 g, 40.7 mmol), and NaHCO₃ (4.33 g,51.5 mmol, in portions) were successively added to distilled, degassedCH₃OH (200 mL). The reaction mixture, pH 6.2-6.6, was heated at 70° C.for 72 h. After cooling to room temperature, the bulk of the solvent wasremoved by rotary evaporation. The residue was dissolved in 8% NaHCO₃(200 mL) and was extracted with CHCl₃ (3×100 mL). The aqueous portionwas cooled in an ice water bath, acidified to pH≈1 with 5 N HCl, andextracted with EtOAc (4×100 mL). The EtOAc extracts were washed withsaturated NaCl and were concentrated in vacuo. Drying under high vacuumfurnished 9.74 g of 9 (90%) as an orange oil: [α]²⁰+61.9°; ¹H NMR (D₂O)δ 1.77 (s, 3H), 3.35 (s, 3H), 3.56-3.62 (m, 3H), 3.64-3.73 (m, 4H),3.75-3.79 (m, 2H), 3.92-3.96 (m, 2H), 3.99 (d, 1H, J=11.6), 4.25-4.31(m, 2H), 6.99 (t, 1H, J=8.2), 7.26-7.33 (m, 2H); ¹³C NMR δ 24.52, 39.93,59.07, 69.04, 69.83, 70.49, 70.64, 70.86, 71.97, 83.21, 116.33, 117.94,118.50, 122.80, 147.67, 150.24, 172.38, 176.10; HRMS m/z calcd forC₁₈H₂₆NO₇S, 400.1429 (M+H). found, 400.1413.

Example 9

Ethyl(S)-4,5-Dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylate(16). Flame activated K₂CO₃ (5.05 g, 36.6 mmol) followed by 15 (11.11 g,34.9 mmol) in acetone (50 mL) were added to 14 (9.35 g, 33.2 mmol) inacetone (300 mL). The reaction mixture was heated at reflux for 3 days.Additional K₂CO₃ (4.59 g, 33.2 mmol) and 15 (2.12 g, 6.65 mmol) inacetone (5 mL) were added, and the reaction mixture was heated at refluxfor 1 day. After cooling to room temperature, solids were filtered andthe solvent was removed by rotary evaporation. The residue was dissolvedin 1:1 0.5 M citric acid/saturated NaCl (320 mL) and was extracted withEtOAc (3×150 mL). The combined organic extracts were washed withdistilled H₂O (200 mL) and saturated NaCl (200 mL) and were concentratedin vacuo. Purification using flash column chromatography eluting with50% EtOAc/petroleum ether generated 12.0 g of 16 (84%) as an oil:[α]²³+40.2 (c 1.09); ¹H NMR δ 1.30 (t, 3H, J=7.2) 1.66 (s, 3H), 3.19 (d,1H, J=11.2), 3.38 (s, 3H), 3.54-3.57 (m, 2H), 3.64-3.70 (m, 4H),3.72-3.76 (m, 2H), 3.81-3.88 (m, 3H), 4.12-4.17 (m, 2H), 4.20-4.28 (m,2H), 6.46 (dd, 1H, J=8.8, 2.4), 6.49 (d, 1H, J=2.4), 7.28 (d, 1H,J=8.4), 12.69 (s, 1H); ¹³C NMR δ 14.21, 24.58, 39.94, 59.17, 62.01,67.65, 69.60, 70.69, 70.76, 70.98, 72.03, 83.22, 101.51, 107.41, 109.98,131.77, 161.27, 163.09, 170.90, 172.95. Anal. (C₂₀H₂₉NO₇S) C; H; N.

Example 10

2-Hydroxy-3-(3,6,9-trioxadecyloxy)benzonitrile (18). Compound 17 (5.3 g,39.2 mmol) was added to a suspension of 60% NaH (3.13 g, 78.2 mmol) inDMSO (60 mL) using oven-dried glassware. After the reaction mixture wasstirred at room temperature for 1 h, 15 (12.49 g, 39.22 mmol) in DMSO(25 mL) was introduced. After 24 h of stirring at room temperature, thereaction mixture was poured with stirring into cold water (100 mL) andwas extracted with CHCl₃ (3×100 mL). The aqueous phase was acidified topH≈1 with 6 N HCl and was extracted with CHCl₃ (5×60 mL). The latterCHCl₃ extracts were concentrated in vacuo. Purification using columnchromatography by gravity eluting with 10% CH₃OH/CHCl₃ gave 7.84 g of 18(70%) as an oil: ¹H NMR δ 3.40 (s, 3H), 3.58-3.62 (m, 2H), 3.65-3.73 (m,4H), 3.75-3.78 (m, 2H), 3.83-3.87 (m, 2H), 4.14-4.18 (m, 2H), 6.79-6.85(m, 1H), 7.09 (dd, 1H, J=7.8, 1.6), 7.15-7.18 (m, 1H), 8.6 (s, 1H); ¹³CNMR δ 57.25, 67.76, 67.85, 68.79, 68.92, 69.06, 70.36, 98.38, 115.44,116.55, 118.51, 123.13, 145.98, 149.46; HRMS m/z calcd for C₁₄H₂₀NO₅,282.134 (M+H). found, 282.135.

The description of the invention herein demonstrates the impact ofintroducing a 3,6,9-trioxadecyloxyl group at various positions of thedesazadesferrithiocin (DADFT) aromatic ring on iron clearance and organdistribution is described. Three DADFT polyethers are evaluated:(S)-4,5-dihydro-2-[2-hydroxy-4-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-4′-(HO)-DADFT-PE, 3],(S)-4,5-dihydro-2-[2-hydroxy-5-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-5′-(HO)-DADFT-PE, 6], and(S)-4,5-dihydro-2-[2-hydroxy-3-(3,6,9-trioxadecyloxy)phenyl]-4-methyl-4-thiazolecarboxylicacid [(S)-3′-(HO)-DADFT-PE, 9]. The iron-clearing efficiency (ICE) inrodents and primates is shown to be very sensitive to which positionalisomer is evaluated, as is the organ distribution in rodents. Thepolyethers had uniformly higher ICEs than their corresponding parentligands in rodents, consistent with in vivo ligand-serum albumin bindingstudies. Ligand 9 is the most active polyether analogue in rodents andis also very effective in primates, suggesting a higher index of successin humans. In addition, this analogue is also shown to clear more ironin the urine of the primates than many of the other chelators.

Having now described a few embodiments of the invention, it should beapparent to those skilled in the art that the foregoing is merelyillustrative and not limiting, having been presented by way of exampleonly. Numerous modifications and other embodiments are within the scopeof one of ordinary skill in the art and are contemplated as fallingwithin the scope of the invention and any equivalent thereto. It can beappreciated that variations to the present invention would be readilyapparent to those skilled in the art, and the present invention isintended to include those alternatives. Further, because numerousmodifications will readily occur to those skilled in the art, it is notdesired to limit the invention to the exact construction and operationillustrated and described, and accordingly, all suitable modificationsand equivalents may be resorted to, falling within the scope of theinvention.

1. A method of treating a pathological condition in a subject comprisingadministering to the subject a therapeutically effective amount of adesazadesferrithiocin analog of formula:

wherein: R₁, R₄, and R₅ are the same or different and are H, straight orbranched chain alkyl having up to 14 carbon atoms, or arylalkyl whereinthe aryl portion is hydrocarbyl, the alkyl portion is straight orbranched chain, and the arylalkyl group has up to 14 carbon atoms; R₂ isH, straight or branched chain alkyl having up to 14 carbon atoms, alkoxyhaving up to 14 carbon atoms, or arylalkyl wherein the aryl portion ishydrocarbyl, the alkyl portion is straight or branched chain, and thearylalkyl group has up to 14 carbon atoms; R₃ is—[(CH₂)_(n)—O]_(x)—[(CH₂)_(n)—O]_(y)-alkyl; n is, independently, aninteger from 1 to 8; x is 3; y is 0; and —OR₃ occupies the 3-position onthe phenyl ring; or a salt, hydrate, or solvate thereof; wherein thepathological condition is metal overload, diabetes, liver disease,Friedreich ataxia (FRDA), heart disease, or radiation injury.
 2. Themethod of claim 1, wherein R₁ is H or —CH₃.
 3. The method of claim 1wherein R₂ is H or —OCH₃.
 4. The method of claim 1, wherein —OR₃ is—O—[(CH₂)₂—O]₃—CH₃.
 5. The method of claim 1, wherein R₄ and R₅ are eachH.
 6. The method of claim 1, wherein R₁ is —CH₃.
 7. The method of claim1, wherein R₂ is H.
 8. The method of claim 1, wherein each instance of nis
 2. 9. The method of claim 1, wherein the salt is an alkali oralkaline earth metal salt.
 10. The method of claim 1, wherein the saltis a sodium salt, potassium salt, magnesium salt, or calcium salt. 11.The method of claim 1, wherein the metal is trivalent.
 12. The method ofclaim 1, wherein the pathological condition is metal overload.
 13. Themethod of claim 12, wherein the metal overload is iron overload.
 14. Themethod of claim 13, wherein the iron overload is primaryhemochromatosis.
 15. The method of claim 13, wherein the iron overloadis secondary hemochromatosis.
 16. The method of claim 13, wherein theiron overload is focal iron overload.
 17. The method of claim 13,wherein the iron overload is thalassemia.
 18. The method of claim 1,wherein the pathological condition is diabetes.
 19. The method of claim1, wherein the pathological condition is liver disease.
 20. The methodof claim 1, wherein the pathological condition is Friedreich ataxia(FRDA).
 21. The method of claim 1, wherein the pathological condition isheart disease.
 22. The method of claim 1, wherein the pathologicalcondition is radiation injury.
 23. A method of treating iron overloaddue to blood transfusions in a subject suffering therefrom comprisingadministering to the subject a therapeutically effective amount of adesazadesferrithiocin analog of formula:

wherein: R₁, R₄, and R₅ are the same or different and are H, straight orbranched chain alkyl having up to 14 carbon atoms, or arylalkyl whereinthe aryl portion is hydrocarbyl, the alkyl portion is straight orbranched chain, and the arylalkyl group has up to 14 carbon atoms; R₂ isH, straight or branched chain alkyl having up to 14 carbon atoms, alkoxyhaving up to 14 carbon atoms, or arylalkyl wherein the aryl portion ishydrocarbyl, the alkyl portion is straight or branched chain, and thearylalkyl group has up to 14 carbon atoms; R₃ is—[(CH₂)_(n)—O]_(x)—[(CH₂)_(n)—O]_(y)-alkyl; n is, independently, aninteger from 1 to 8; x is 3; y is 0; and —OR₃ occupies the 3-position onthe phenyl ring; or a salt, hydrate, or solvate thereof.
 24. The methodof claim 23, wherein the analog is of the formula:

or a salt, hydrate, or solvate thereof.
 25. The method of reducingoxidative stress in a subject comprising administering to the subject atherapeutically effective amount of a desazadesferrithiocin analog offormula:

wherein: R₁, R₄, and R₅ are the same or different and are H, straight orbranched chain alkyl having up to 14 carbon atoms, or arylalkyl whereinthe aryl portion is hydrocarbyl, the alkyl portion is straight orbranched chain, and the arylalkyl group has up to 14 carbon atoms; R₂ isH, straight or branched chain alkyl having up to 14 carbon atoms, alkoxyhaving up to 14 carbon atoms, or arylalkyl wherein the aryl portion ishydrocarbyl, the alkyl portion is straight or branched chain, and thearylalkyl group has up to 14 carbon atoms; R₃ is—[(CH₂)_(n)—O]_(x)—[(CH₂)_(n)—O]_(y)-alkyl; n is, independently, aninteger from 1 to 8; x is 3; y is 0; and —OR₃ occupies the 3-position onthe phenyl ring; or a salt, hydrate, or solvate thereof.